WO2023130076A2

WO2023130076A2 - Targets and pathways for the production of alkaloidal compounds

Info

Publication number: WO2023130076A2
Application number: PCT/US2022/082632
Authority: WO
Inventors: Thomas Henley; Modassir CHOUDHRY; Jose FERNANDEZ-GOMEZ; Antoine LARRIEU; Beata ORMAN-LIGEZA; Umar Mohammed; Emma MCKECHNIE-WELSH
Original assignee: Empyrean Neuroscience Inc
Current assignee: Empyrean Neuroscience Inc
Priority date: 2021-12-31
Filing date: 2022-12-30
Publication date: 2023-07-06
Anticipated expiration: 2024-06-30
Also published as: EP4457336A2; US12104179B2; US20240101976A1; WO2023130076A3; WO2023130075A2; WO2023130078A2; WO2023130075A3; WO2023130078A3; CN118984868A; US20250171749A1

Abstract

This disclosure relates generally to non-naturally organisms, as well as compositions and methods thereof, which host biosynthetic pathways that can produce novel or rare alkaloids. In some embodiments, the non-naturally occurring organism are fungi from division Basidiomycota.

Description

TARGETS AND PATHWAYS FOR THE PRODUCTION OF ALKALOIDAL COMPOUNDS

CROSS REFERENCE TO RELATED APPLICATIONS

[1] The present application claims priority to U.S. Provisional Application No. 63/295,735, filed December 31, 2021, U.S. Provisional Application No. 63/295,739, filed December 31, 2021, U.S. Provisional Application No. 63/295,742, filed December 31, 2021, and U.S. Provisional Application No. 63/295,723, filed December 31, 2021, the entire contents of each of which are hereby incorporated by reference in their entirety.

BACKGROUND

[2] Nearly one in five adults living in the U.S. suffer from a mental health disorder, which can come, for example, in the form of severe depression, anxiety, obsessive-compulsive disorder, addiction, or substance abuse. There is no absolute cure for these conditions, so there is an increased importance for continued care and symptom management. Titus, there is a need for innovative therapeutic solutions.

SUMMARY

[3] This disclosure relates to non-naturally occurring organisms, as well as compositions and methods thereof, hosting biosynthesis pathways that produce novel or rare alkaloids. The disclosure further relates to modulating said biosynthesis pathways for the production, increased production, or decreased production of bioactive alkaloidal compounds.

[4] In some embodiments is provided an engineered fungal cell comprising a heterologous PsiL gene. In some embodiments, the heterologous PsiL gene comprises a nucleotide sequence that is at least 95% identical to SEQ ID NOS: 322. In some embodiments, the heterologous PsiL gene comprises a sequence that has 100% identity to SEQ ID NO: 322. In some embodiments, the engineered fungal cell is comprised in a multicellular organism. In some embodiments, the multicellular organism is from division Basidiomycota. In some embodiments, the multicellular organism is a species selected from the group consisting of: Psilocybe cyanescens, Psilocybe azurescence, and Psilocybe tampanensis. In some embodiments, the multicellular organism is the species Psilocybe cubensis. In some embodiments, the engineered fungal cell comprises an alkaloid. In some embodiments, the alkaloid is selected from the group consisting of: N,N- dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-Z-tryptophan, 5-hydroxy-/,- tryptophan, 7-hydroxy-Z-tryptophan, isonorbaeocystin, 4-phosporyloxy-N,N-dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium, 4- phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3,4- Methylenedioxymethamphetamine, harmine, harmane, a P-carboline, and a salt of any of these. In some embodiments, the alkaloid is selected from the group consisting of: psilocybin, psilocin, norpsilocin, norbaeocystin, Baeocystin, aeruginascin, psilocin-dlO, psilocybin-d4, N-acetyl- N,N- dimethytryptamine, 4-hydroxy-Z-tryptophan, 5 -hydroxy -/.-tryptophan, 7-hydroxy-Z-tryptophan, isonorbaeocystin, 4-phosporyloxy-N,N-dimethyltryptamine, serotonin, aeruginascin, 2-(4- Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium, 4-phosphoryloxy-N,N- dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, harmine, harmane, a P-carboline, and a salt of any of these. In some embodiments, the engineered fungal cell comprises an alkaloid that has an increased expression of an alkaloid as compared to an expression of the alkaloid in a comparable fungal cell without a heterologous PsiL gene.

[5] In some embodiments, are provided methods of overexpressing a gene involved in psilocybin biosynthesis in a psilocybe protoplast, the methods comprising: targeting a gene involved in psilocybin biosynthesis locus through homologous directed repair, wherein a selfreplicating plasmid is transfected into a psilocybe protoplast; and introducing a B-AMA1 replication origin sequence into the self-replicating plasmid of the psilocybe protoplast, wherein the self-replicating plasmid is not integrated into the genome of the protoplast; thereby overexpressing the gene in the psilocybe protoplast. In some embodiments, the plasmid is not integrated into the genome of the fungal protoplast. In some embodiments, the gene contained within the plasmid is integrated into the genome of the psilocybe protoplast, however, the plasmid is not integrated. In some embodiments, the psilocybe protoplast is a psilocybe cubensis protoplast. In some embodiments, the gene involved in psilocybin biosynthesis is a PsiD gene. In some embodiments, the PsiD gene has at least about 85% identity to SEQ ID NO: 120. In some embodiments, the PsiD gene has at least 95% identity to SEQ ID NO: 120. In some embodiments, the PsiD gene has 85% identity to SEQ ID NO: 120. In some embodiments, the PsiD gene has 95% identity to SEQ ID NO: 120. In some embodiments, the self-replicating plasmid comprises a hygromycin resistance cassette. In some embodiments, the hygromycin resistance gene after introduction into the protoplast is subsequently spontaneously removed optionally without selection pressure. In some embodiments, the B-AMA1 replication origin plasmid comprises an spCas sequence. In some embodiments, the B-AMA1 replication origin sequences, the spCas sequence, or a combination thereof comprises 95% identity to any one of SEQ ID NOs: 640-647. In some embodiments, the B-AMA1 sequence comprises 95% identity to SEQ ID NO: 648 or 649. [6] In one aspect, this disclosure provides a composition comprising a genetically modified organism, wherein the genetically modified organism has an exogenous nucleic acid encoding a heterologous gene from the Psilocybe gene cluster, which, when expressed, produces an alkaloid. In some embodiments, the heterologous gene encodes a tryptamine monooxygenase. In some embodiments, the heterologous gene encodes a methyl transferase. In some embodiments, the heterologous gene encodes a helix-loop-helix transcription factor.

[7] In another aspect, this disclosure provides a composition comprising a genetically modified organism, wherein the genetically modified organism comprises a genetic modification involving a heterologous PsiH2 gene encoding a tryptamine monooxygenase, wherein the tryptamine monooxygenase is expressed in an amount sufficient to produce an alkaloid or a precursor to the alkaloid.

[8] In another aspect, this disclosure provides a composition comprising a genetically modified organism comprising an exogenous nucleic acid, wherein the exogenous nucleic acid encodes a heterologous enzyme that is not expressed in a detectable amount in a comparable organism without said genetic modification. In some embodiments, the heterologous enzyme is a tryptamine monooxygenase. In some embodiments, the heterologous enzyme is a methyl transferase. In some embodiments, the exogenous nucleic acid encodes the heterologous tryptamine monooxygenase and the heterologous methyl transferase.

[9] This disclosure further provides compositions and methods for genetically modifying organisms to increase the production of new or rare alkaloid(s).

[10] In one aspect, this disclosure provides a gene editing system for enhanced expression of a psychotropic alkaloid in a fungal cell. The system comprises an endonuclease and at least one guide polynucleotide, or one or more nucleic acids encoding the endonuclease and the at least one guide polynucleotide, wherein the guide polynucleotide binds to a nucleic acid that encodes or regulates a gene that modulates production of an alkaloid, optionally psychotropic alkaloid, in a fungal cell. The system further comprises a reagent that increases incorporation of the endonuclease or the at least one guide polynucleotide, or the one or more nucleic acids encoding the endonuclease or the at least one guide polynucleotide, into the fungal cell as compared to the incorporation without the reagent. In some embodiments, the fungal cell is a fungal protoplast. In some embodiments, the system comprises one or more nucleic acids encoding the gene editing system can be introduced into the protoplast. In some embodiments, the one or more nucleic acids comprise non-replicating DNA. In some embodiments, the system comprises the endonuclease and the at least one guide polynucleotide in the format of an active ribonucleoprotein. In some embodiments, the reagent comprises a nonionic surfactant, a lipid nanoparticle, or an agent that depolymerizes microtubules. In some embodiments, the reagent comprises:

some embodiments the reagent has a molecular mass of

647 grams/mole.

[11] In another aspect, this disclosure provides gene editing systems for genetically modifying a fungal cell, wherein the system comprises an endonuclease and at least one guide polynucleotide, or one or more nucleic acids encoding said endonuclease and the at least one guide polynucleotide, wherein the guide polynucleotide comprises a sequence that can bind to a gene that comprises a sequence comprising one of SEQ ID NOS: 60-116, wherein, binding of the guide polynucleotide to the gene in a fungal cell leads to a genetic modification that modulates production of one or more alkaloids. In some embodiments, the guide polynucleotide comprises a sequence that is complementary to an alkaloid synthase gene. In some embodiments, the guide polynucleotide binds to one of the genes listed in Table 1 or in Table 2. In some embodiments, the guide polynucleotide binds to one of the genes listed in Table 1, Table 2 or Tables 3A-3B.

[12] In some embodiments, the gene editing system comprises a promoter, which can be operably linked to the heterologous gene comprised in the gene editing system. In another aspect, this disclosure provides a gene editing system for genetically modifying a fungal cell, the system comprising at least one nucleic acid sequence encoding a guide polynucleotide that binds to a nucleic acid involved in expression or regulation of a psychotropic alkaloid, wherein the at least one nucleic acid sequence is operably linked to a first gene promoter, and a nucleic acid sequence encoding an endonuclease operably linked to a second gene promoter, wherein the second gene promoter is distinct from the first gene promoter, wherein when the gene editing system is expressed in the fungal cell, the gene editing system introduces a genetic modification into the genome of the fungal cell. The first gene promoter and the second gene promoter can have different promoter activities. In some embodiments, the first gene protomer is a U6 gene promoter. In some embodiments, the second gene promoter is a GDP gene promoter.

[13] In another aspect, this disclosure provides a gene editing system for multiplex gene engineering of a fungal cell, the system comprising a vector encoding at least two guide polynucleotides that each bind to a nucleic acid that encodes or regulates a gene that modulates production of a psychotropic alkaloid in a fungal cell, and an endonuclease. When the at least two guide polynucleotides and the endonuclease are expressed in a fungal cell, the at least two guide polynucleotides and the endonuclease introduce a genetic modification into the genome of the fungal cell that modulates expression of an alkaloid. In some embodiments, the vector is a bacterial vector. In some embodiments, the vector comprises border sequences that facilitates the incorporation of at least a portion of the vector into the fungal cell by a bacterium.

[14] In another aspect, this disclosure provides a kit comprising a gene editing system as described herein for genetically modifying a fungal cell. The kit can include reagents for delivering the gene editing system into the fungal cell. The kit may also include instructions.

[15] In another aspect, this disclosure provides a method for genetically modifying a fungal cell. The method includes introducing a gene editing system as described herein into a fungal cell. In some embodiments, the method further includes expressing the gene editing system inside the fungal cell, wherein expression of the gene editing system inside the fungal cell results in a genetic modification that leads to the increased production of one or more psychotropic alkaloids as compared to a comparable fungal cell devoid of said gene editing system.

[16] In another aspect, this disclosure provides a composition comprising an alkaloid according

R² is H, -OH, -0P(0)(0M)₂, -C(O)CH₃, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

R³ is H, -OH, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

R⁴ and R⁵ are each independently H, -OH, or -OCH₃;

R^A is H, -COOH;

R^x, R^y, and R^z are each independently H, -CH₃, -C(O)CH₃, -CH₂CH₃, or -CH(CH₃)₂; and M is H, Li⁺, Na⁺, K⁺ or is absent, and further comprising material from a ruptured fungal cell.

[17] In some embodiments, the material from the ruptured fungal cell includes a nucleic acid. In some embodiments, the nucleic acid encodes a methyl transferase. In some embodiments, the nucleic acid encodes a tryptamine monooxygenase. In some embodiments, the nucleic acid encodes a helix-loop-helix transcription factor.

[18] In another aspect, this disclosure provides a composition comprising a non-naturally occurring fungal cell. The fungal cell includes a genetic modification that results in the fungal cell expressing a heterologous tryptamine monooxygenase, or a heterologous methyltransferase, that is not expressed in a detectable amount in a comparable fungal cell without the genetic modification. In some embodiments, the fungal cell comprises the heterologous tryptamine monooxygenase. In some embodiments, the fungal cell comprises the heterologous methyltransferase. In some embodiments, the fungal cell comprises the tryptamine monooxygenase and the heterologous methyltransferase.

[19] In another aspect, this disclosure provides a composition comprises a genetically modified fungal cell comprising a genetic modification that comprises an exogenous nucleic acid encoding a helix-loop-helix transcription factor that binds to a regulatory element of one or more genes associated with an alkaloid biosynthesis pathway, wherein the exogenous nucleic acid comprises a sequence that has at least 95% identity to one of SEQ ID NO: 7 or 14.

[20] In another aspect, this disclosure provides a genetically modified fungal cell comprising a first genetic modification that results in an increased expression of a heterologous tryptamine monooxygenase as compared to a comparable fungal cell without the first genetic modification, and a second genetic modification that results in an increased expression of a helix-loop-helix transcription factor as compared to a comparable fungal cell without the second genetic modification, wherein the helix-loop-helix transcription factor binds to a regulatory element of one or more genes that encode products of an alkaloid biosynthesis pathway. In some embodiments, the helix-loop-helix transcription factor binds to the regulatory element of one or more genes to thereby upregulate the expression of those genes inside the fungal cell.

[21] In another aspect, this disclosure provides a method of producing an alkaloid. The method comprises introducing a genetic modification into a fungal cell, wherein the genetic modification comprises an exogenous nucleic acid that encodes a tryptamine monooxygenase, or a methyltransferase, that is heterologous to the fungal cell. The method further includes expressing the heterologous tryptamine monooxygenase, or the heterologous methyltransferase, in the fungal cell to produce the alkaloid. In some embodiments, the exogenous nucleic acid encodes the heterologous tryptamine monooxygenase. In some embodiments, the exogenous nucleic acid encodes the heterologous methyl transferase. In some embodiments, the exogenous nucleic acid encodes the heterologous tryptamine monooxygenase and the heterologous methyl transferase. [22] In another aspect, this disclosure provides a method of producing an alkaloid, the method comprising introducing a genetic modification into a fungal cell, wherein the genetic modification comprises an exogenous nucleic acid that encodes a tryptamine monooxygenase that is heterologous to the fungal cell expressing the tryptamine monooxygenase in the fungal cell to produce the alkaloid; and wherein the alkaloid comprises a compound of Formula (I):

a tautomer thereof, or a pharmaceutically acceptable salts of any of the foregoing wherein:

R¹ is H or -CH₃;

R² is H, -OH, -OP(O)(OM)₂, -C(O)CH₃, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

R³ is H, -OH, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

R⁴ and R⁵ are each independently H, -OH, or -OCH₃;

R^A is H, -COOH;

R^x, R^y, and R^z are each independently H, -CH₃, -C(O)CH₃, -CH₂CH₃, or -CH(CH₃)₂; and M is H, Li⁺, Na⁺, K⁺ or is absent.

BRIEF DESCRIPTION OF THE DRAWINGS

[23] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure can be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[24] FIG. 1 illustrates an alkaloid biosynthesis pathway of a genetically modified organism with a genetic modification that upregulates expression of tryptophan decarboxylase.

[25] FIG. 2 illustrates a biosynthesis pathway hosted by an organism engineered for producing psilocybin. The organism includes a genetic modification that suppresses psilocybin phosphatase.

[26] FIG. 3 illustrates an additional alkaloid biosynthesis pathway of a genetically modified organism. The organism includes a genetic modification that upregulates expression of tryptophan decarboxylase and a genetic modification that downregulates expression of psilocybin phosphatase.

[27] FIG. 4 shows a sequence alignment comparing PsiH and PsiH2 gene products from four different psilocybin-producing fungi.

[28] FIG. 5 shows a phylogenetic tree generated for PsiH and PsiH2 genes from four psilocybin producing fungi.

[29] FIG. 6 shows an optimized pl300Cambia plasmid encoding PsiD.

[30] FIG. 7 shows a PCR gel confirming a PsiD genetic modification of a fungal cell. “C+” indicates a lane loaded with a positive control. “C-” indicates a lane loaded with a negative control, “wt” indicates a lane from a PCR run performed on wild-type fungal material. The absence of signal in the “wt” lane and positive signal in lanes 1-16 demonstrates a PsiD transgene is integrated into the genome of the fungal cell.

[31] FIG. 8 shows an image capture of electrophoresis gels confirming PsiD upregulation in transgenic mycelia. The gels are from cDNA analyses of PsiD mRNA expression in my celia transformed with the GT6 plasmid. The top gel (601) is from a cDNA analysis (RT-PCR) of total expressed mRNA in transgenic mycelia. The bottom gel (603) is from a cDNA analysis of expressed PsiD mRNA in transgenic mycelia in which no reverse transcriptase was added during the RT-PCR assay, thus confirming the PsiD signal observed in the top gel is expressed mRNA and not contaminating DNA.

[32] FIG. 9 is an image of non-transgenic wild-type mycelia (901) and transgenic mycelia (903) that express elevated levels of PsiD.

[33] FIG. 10 shows a transgenic mycelial mass (1004) upon primordia formation (1005) in comparison with a wild type mycelial mass (1003).

[34] FIG. 11 shows an image of a side-by-side comparison of a PsiD transgenic fungus compared to a wild-type fungus.

[35] FIG. 12 illustrates a biosynthesis pathway. The pathway shows alkaloids that are upregulated upon increased expression of PsiD.

[36] FIG. 13A-13D show concentrations of alkaloids measured in PsiD transgenic and wildtype fungi as measured by LC-MS. The Y-axis shows area counts as detected by the LC-MS. The X-axis identifies samples of genetically modified fungi with increased expression of PsiD.

[37] FIG. 14 shows the content of psilocybin and psilocin in PsiD transgenic fungi as compared with wild-type fungi as measured by LC-MS. [38] FIG. 15 shows amounts of certain alkaloids measured in transgenic and wild-type fungi by LC-MS. The Y-axis shows area counts as detected by the LC-MS. The X-axis identifies samples.

[39] FIG. 16 shows the content of psilocybin and psilocin in the PsiD transgenic fungi as compared with wild-type fungi as measured by LC-MS.

[40] FIG. 17 illustrates certain alkaloids that are formed from psilocin.

[41] FIGS. 18A-18B show LC-MS data on quinoid and quinoid dimers related to psilocin. Samples 1771 and 1772 are from transgenic fungi. Sample 1773 is from a comparable wild type control. FIG. 18A shows enzymatically produced sample data. FIG. 18B show ESI produced data.

[42] FIG. 19A-19D shows relative amounts of alkaloids in PsiD transgenic fungi as compared with wild-type fungi. The Y axis shows area counts as detected by the LC-MS. The X-axis identifies samples. Samples 1771 and 1772 are from transgenic fungi. Sample 1773 is from a comparable wild type control. FIG. 19A shows relative amounts of 4-hydroxytryptamine. FIG. 19B shows relative amounts of 4-hydroxytrimethyltryptamine. FIG. 19C shows relative amounts of aeruginascin. FIG. 19D is a chart showing data of FIGS. 19A-19C.

[43] FIG. 20 shows an illustration of genes from the psilocybin cluster from six psilocybin- producing fungal species. The genes are color coded in grayscale according to the annotated key.

[44] FIGs. 21A-21C shows P-carbolines biosynthesis pathways of bacteria (FIG. 21 A) and plants (FIG. 21B). FIG. 21C shows biosynthetic pathways metabolizing L-tryptophan to various alkaloids including, for example, N,N-dimethyltryptophan, using AADC (also referred to herein, as AAAD), and INMT gene products.

[45] FIG. 22 illustrates methyl transfer steps during biosynthesis of N,N-dimethyl-L- tryptophan (DMT) and psilocybin by TrpM and PsiM, respectively.

[46] FIG. 23 shows a comparison of PsiM gene product alignment for four different psilocybin-producing psilocybe fungal species.

[47] FIG. 24 is a schematic representation of a cloning system used for genetic engineering.

[48] FIG. 25 shows an alignment of three U6 promoters used in the cloning system.

[49] FIG. 26 shows an illustration on guide oligo design.

[50] FIG. 27A-27B show a sequence alignment comparing PsiH and PsiH2 gene products from four different psilocybin-producing fungi. The arrow shown on FIG. 28B shows alignment for the P. Cyanescense PsiH2 gene in comparison with other psilocybe fungi species.

[51] FIG. 28A-28F shows analytical analyses of alkaloids produced in genetically engineered fungi for two strains (39.9 and 39.6) with analyses run in triplicate. [52] FIG. 29A-29B show graphical representations of PcINMT expression using a PcINMT plasmid optimized for Psilocybe cubensis in 24 independent cell lines transformed in genetically engineered mycelium. Expression was measured in arbitrary units.

[53] FIG. 30A-30B show an image of a molecular ladder for Psilocybe cubensis INMT (FIG 30A) FIG 30B shows a graphical representation of PcINMT expression in selected transgene copy lines.

[54] FIG. 31A-31C show representations of TrpM expression using genetically engineered fungi comprising various primer sequences. FIG. 31A shows an image of a molecular ladder for TrpM. FIGs. 31B-31C show TrpM expression in selected transgene copy lines. TrpM-03, for example, comprised multiple transgene copies.

DETAILED DESCRIPTION

[55] The following discussion of the present disclosure has been presented for purposes of illustration and description. The following is not intended to limit the disclosure to the form or forms discussed herein. Although the description of the present disclosure has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

[56] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[57] Although various features of the disclosure may be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the disclosure may be described herein in the context of separate embodiments for clarity, various aspects and embodiments can be implemented in a single embodiment.

[58] Any gene described herein may independently have a percentage sequence identity of about: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Any gene described herein may independently have a percentage sequence identity of up to: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Any gene described herein may independently have a percentage sequence identity of at least: 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9%. [59] This disclosure relates generally non-naturally occurring organisms, as well as compositions and methods thereof, that are useful for producing new or rare alkaloids. In certain aspects, this disclosure relates to organisms that are genetically modified to possess markedly different characteristics as compared to comparable wild-type organisms found in nature. As such, the genetically modified organisms of this disclosure are not naturally occurring.

[60] The non-naturally occurring organisms described herein are, by way of one or more genetic modifications, useful for producing new or rare alkaloids with desirable properties. The one or more genetic modifications can result in an organism hosting a new biosynthetic pathway or an enhanced biosynthetic pathway that results in the production of the one or more desired alkaloids. In some embodiments, the non-naturally occurring organisms described herein produce one or more desired alkaloids that that do not exist in nature. In some embodiments, the non-naturally occurring organisms described herein produce one or more desired alkaloids that in an amount that cannot be found in comparable organisms found in nature. In some embodiments, the non-naturally occurring organisms described herein produce at least two desired alkaloids in a combination that cannot be found in nature.

[61] Provided herein are non-naturally occurring organisms, as well as compositions and methods thereof, which possess biochemical phenotypes that are rich in desirable compounds, including desirable combinations of those compounds. This disclosure also provides methods to modulate production of specific compounds and combinations of those compounds within the genetically modified organism. For example, in some embodiments this disclosure takes advantage of gene editing systems to make genetic modifications that upregulate or downregulate expression of one or more compounds with high specificity. Accordingly, methods described herein can be used to tailor biosynthetic pathways of organisms to produce genetically modified organisms that can produce increased quantities of certain compounds, including new compounds, that are of interest. In some embodiments, methods of this disclosure make use of gene editing systems employing nucleic acid guided nucleases such that one or more desired genetic modifications can be made in a tailored and controlled manner. Compositions and methods described herein also provide for efficient production of alkaloids, allowing for scale up of production method to an industrial scale. For example, the production of one or more alkaloids in a genetically modified organism described herein can make use of large-scale bioreactors or production systems to provide a consistent, economical, and high level of production. Moreover, alkaloids produced by the methods described herein can be used or formulated into selected compositions, such as a pharmaceutical composition, and even provided in single dose format. [62] This disclosure provides gene editing systems, compositions, and methods for genetically modifying organisms for the production of alkaloids. This disclosure provides gene editing systems that, when expressed in an organism, result in a genetic modification that leads to the increased production of one or more desired alkaloids. The result of the genetic modification introduced by a composition or a method described herein is a genetically modified organism that possess a markedly different characteristics as compared to comparable wild-type organism found in nature. As such, compositions and methods of this disclosure can produce genetically modified organisms that are not naturally occurring. The non-naturally occurring genetically modified organisms described herein are, by way of one or more genetic modifications, useful for producing alkaloids with desirable properties. In particular, the compositions and methods described herein can produce genetically modified organisms with new or enhanced biosynthetic pathways that lead to the production, or the increased production, of the one or more desired alkaloids. The one or more desired alkaloids as described herein can be new to nature or produced in quantities that cannot be found in comparable wild-type organisms.

[63] This disclosure further relates to gene editing systems and methods that can precisely modify genetic material in eukaryotic cells, which enables a wide range of high value applications in medical, pharmaceutical, drug discovery, agricultural, basic research and other fields. Fundamentally, the genome editing systems and methods provided herein enable the capability to introduce, or remove, one or more nucleotides at specific locations in eukaryotic genomes. The genome editing systems also allows for the ability to incorporate exogenous nucleic acids, sometimes referred to as a donor sequence, into an organism for expression by the organism. Accordingly, this disclosure provides gene editing systems and methods thereof that allow for targeted edits, such as deleting, inserting, mutating, or substituting specific nucleic acid sequences, of an organism to produce a genetically modified organism. Organisms genetically modified by a gene editing system, composition, or method described herein can provide a source of a new or rare drug such as one or more fungal derived alkaloids. The organism can be a fungal cell from division Basidiomycota. The fungal cell can be a fungal protoplast.

[64] This disclosure further relates to compositions and methods for genome engineering with a gene-editing system, for example a CRISPR enzyme-based gene editing system. This disclosure provides compositions and methods useful to genetically modify an organism, such as, a fungal protoplast, with a gene editing system. In some embodiments, this disclosure relates to the discovery that Cas endonucleases can be used in the fungal kingdom in combination with guide polynucleotides and donor sequences to provide a toolbox of options from which to pick and choose genetic modifications that can result in genetically modified biosynthetic pathways that produce new or rare alkaloidal compounds. In some embodiments, this disclosure provides codon optimized tools for genetically modifying a fungal cell with a CRISPR system. In some embodiments, the alkaloids produced are new to nature alkaloids with significant clinical value.

[65] In some embodiments, this disclosure provides a platform of compositions and methods involving gene editing systems which have particular applicability to organisms that already possess biosynthetic pathways for producing clinically relevant compounds. Accordingly, in some embodiments, this disclosure provides a drug discovery platform that can produce genetic modifications resulting in altered biosynthetic pathways that lead to the production of new compounds, which is useful for drug discovery.

[66] This disclosure also provides certain sequences useful for targeting a polynucleotide guided endonuclease. The sequences can be used to design guide polynucleotides that target a gene editing system to a gene involved in alkaloid production for editing. The targeted edits described herein can be used to create a new biosynthetic pathway within the organism that produces one or more desired alkaloids. For example, the biosynthetic pathway can be engineered with the gene editing system to produce elevated amounts of a desired alkaloid or desired combinations of alkaloids.

[67] The alkaloids produced by genetically modified organism described herein may be desired for their beneficial biological properties. For example, without limiting the scope of the disclosure, a desired alkaloid may exhibit antiproliferation, antibacterial, antiviral, anticancer, insecticidal, antimetastatic, or anti-inflammatory effects. Accordingly, a genetically modified organism produced by methods described herein can possess a biosynthetic pathway that results in the production of alkaloids with known or suspected beneficial properties. In some embodiments, the desired alkaloids compounds can include tryptophan derived compounds. In some embodiments, the alkaloids can include tryptamine-derived compounds.

[68] This disclosure also provides compositions and methods having gene editing systems that can be used on fungal cells to produce a genetically modified fungal cell possessing a biochemical phenotype that is rich in one or more desirable alkaloids. This disclosure also provides compositions and methods to modulate production of specific alkaloids within the genetically modified fungal cell. For example, methods described herein can be implemented to upregulate or downregulate production of one or more alkaloids with high specificity by virtue of one or more nucleic acid guided gene editing systems. Accordingly, methods described herein can be used to tailor biosynthetic pathways of fungal cells to produce genetically modified fungal cells that can produce increased quantities of certain alkaloids of interest. Because the production is carried out in a genetically modified fungal cell, a production method is provided which can be optimized, tailored, and controlled in any desired manner.

DEFINITIONS

[69] As used herein, an “alkaloid” and a “psychotropic alkaloid” refers to any one of a class of nitrogenous organic compounds of plant or fungal origin which have a physiological effect on a subject, such as a human subject. Exemplary alkaloids include psilocybin, psilocin, norpsilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, baeocystin, norbaeocystin, serotonin, melatonin, melanin, N-acetyl-hydroxytryptamine, 4-hydroxy-L-tryptophan, 5-hydroxy-L- tryptophan, 7-hydroxy-L-tryptophan, 4-phosporyloxy-N,N-dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium, 4-phosphoryloxy- N,N-dimethytryptamine, ketamine, normelatonin, 3, 4-m ethylenedi oxymethamphetamine, P- carboline, or any derivative or any analogue thereof.

[70] As used herein, a “biosynthetic pathway” refers to a multi-step, enzyme-catalyzed process whereby a substrate is converted into a compound in a living organism, such as a genetically modified organism. In biosynthesis, e.g., alkaloid biosynthesis, the substrate can be modified, in some instances, converted into another compound, such as a tryptophan derived alkaloid, via a biosynthetic pathway.

[71] As used herein, “Cas9” refers to an RNA guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and/or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat) associated nuclease. An exemplary Cas9, is Streptococcus pyogenes Cas9 (spCas9).

[72] As used herein, “disease” or “disorder” is meant any condition that damages or interferes with the normal function of a cell, tissue, or organ. Exemplary disorders include severe anxiety and addiction.

[73] As used herein, a “cell” refers to a biological cell. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaea cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant, an algal cell, a fungal cell, a fungal protoplast cell, an animal cell, and the like. Sometimes a cell is not originating from a natural organism, e.g., a cell can be a synthetically made, sometimes termed an artificial cell.

[74] The term “effective amount” or “therapeutically effective amount” refers to an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount can include an amount that when administered to a subject can reduce a symptom of a disease or condition or disorder, such as a mental health disorder. In other instances, an effective amount can include an amount that when administered to a subject prevents an unwanted disease or condition or disorder, such as a mental health disorder.

[75] The term “exogenous nucleic acid”, “exogenous nucleic acid sequence”, or “exogenous polynucleotide” refers to a nucleic acid, gene, or genetic material that was transferred into a cell, organism, or animal that originated outside of the cell, organism, or animal. An exogenous nucleic acid can be synthetically produced. An exogenous nucleic acid can be naturally produced, for example, from a different organism of the same species or from a different organism of a different species. An exogenous nucleic acid can be another copy of a nucleic acid that is similar to an endogenous nucleic acid into which the exogenous nucleic acid is incorporated.

[76] As used herein, an “excipient” includes functional and non-functional ingredients in a pharmaceutical composition. The excipient can be an inactive substance that serves as a vehicle or medium for an alkaloid or other compound disclosed herein.

[77] As used herein, “expression” includes any step involved in the production of a polypeptide in a host cell or organism including, but not limited to, transcription, translation, post-translational modification, secretion, or a process by which information from a nucleic acid is used in the synthesis of a functional gene product that enables production of an end product.

[78] The term “functional mushroom,” as used herein, refers to fungal species, derivatives, extracts, and mixtures thereof that have nutritional and/or health benefits. Functional mushrooms include medicinal mushrooms, and adaptogenic mushrooms. Examples of functional mushrooms include, but are not limited to, reishi mushroom, and lion’s mane mushroom.

[79] The term “gene,” as used herein, refers to a nucleic acid (e.g., DNA, such as genomic DNA and cDNA) and its corresponding nucleotide sequence that encodes a gene product, such as an RNA transcript. The term as used herein with reference to genomic DNA can include intervening, non-coding regions as well as regulatory regions. In some uses, the term encompasses the transcribed sequences, including 5 and 3 untranslated regions (5’- TR and 3’-UTR), exons and introns and some genes, the transcribed region can contain “open reading frames” that encode polypeptides. In some uses of the term, a “gene” comprises only the coding sequences (e.g., an “open reading frame” or “coding region”) necessary for encoding a polypeptide. In some cases, genes do not encode a polypeptide, for example, ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. In some cases, the term “gene” includes not only the transcribed sequences, but in addition, also includes non-transcribed regions including upstream and downstream regulatory regions, enhancers and promoters A gene can refer to an “endogenous gene” or a native gene in its natural location in the genome of an organism. A gene can refer to an “exogenous gene” or a non-native gene.

[80] The term “gene editing” and its grammatical equivalents as used herein refers to a genetic engineering method or a genetic modification in which one or more nucleotides are inserted, replaced, or removed from a genome of a cell or organism. For example, gene editing can be performed using a nuclease (e.g., a natural-existing nuclease or an artificially engineered nuclease).

[81] The term “gene knock-out” or “knock-out” refers to any genetic modification that reduces the expression of the gene being “knocked out.” Reduced expression can include no expression. The genetic modification can include a genomic disruption.

[82] The term “genetically modified”, “genetically engineered”, “transgenic”, “genetic modification,” “non-naturally occurring,” and its grammatical equivalents as used herein refer to one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism’s genome. For example, genetic modification can refer to alterations, additions, and/or deletion of one or more genes. A genetically modified cell can also refer to a cell with an added, deleted and/or altered gene.

[83] The term “genetic disruption” or “disrupting”, and its grammatical equivalents as used herein refer to a process of altering a gene, e.g., by deletion, insertion, mutation, rearrangement, or any combination thereof. For example, a gene can be disrupted by knockout. Disrupting a gene can be partially reducing or completely suppressing expression e.g., mRNA and/or protein expression) of the gene. Disrupting can also include inhibitory technology, such as shRNA, siRNA, microRNA, dominant negative, or any other means to inhibit functionality or expression of a gene or protein.

[84] As used herein, “guide RNA” or “gRNA” refers to a polynucleotide which is specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpfl).

[85] The term “nucleotide,” as used herein, generally refers to a base-sugar-phosphate combination. A nucleotide can be composed of three subunit molecules: a nucleobase, a five- carbon sugar (ribose or deoxyribose), and a phosphate group consisting of one to three phosphates. The four nucleobases in naturally occurring DNA include guanine, adenine, cytosine and thymine; in RNA, uracil is used in place of thymine. A nucleotide can comprise a synthetic nucleotide. A nucleotide can comprise a synthetic nucleotide analog. Nucleotides can be monomeric units of a nucleic acid sequence (e.g., deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)). [86] The term “phenotype” and its grammatical equivalents as used herein refer to a composite of an organism’s observable characteristics or traits, such as its morphology, development, biochemical or physiological properties, phenology, behavior, and products of behavior. Depending on the context, the term “phenotype” can sometimes refer to a composite of a population’s observable characteristics or traits.

[87] As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant. A class of plant that can be used in the present disclosure can be generally, as broad as the class of higher and lower plants amenable to mutagenesis including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, fems and multicellular algae. Thus, “plant” includes dicot and monocot plants.

[88] As used herein, “protoplast”: refers to an isolated cell whose cell wall has been removed. A protoplast can refer to an isolated cell in which the cell wall has been removed. The cell wall can be removed by tripping, weakening, creating gaps in, or removing the cell wall, from a plant, bacterial, or fungal cell by mechanical, chemical, or enzymatic means.

[89] As used herein, the terms “protein”, “peptide” and “polypeptide” are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, “peptide” and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein”, “peptide” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.

[90] The term “transgene” refers to a gene or genetic material that is transferred into an organism. For example, a transgene can be a stretch or a contiguous segment of nucleic acid encoding a gene product that is artificially introduced into an organism. The gene or genetic material can be from a different species. The gene or genetic material can be synthetic. When a transgene is transferred into an organism, the organism can then be referred to as a transgenic organism. A transgene can retain its ability to produce RNA or polypeptides (e.g., proteins) in a transgenic organism. A transgene can comprise a polynucleotide encoding a protein or a fragment (e.g., a functional fragment) thereof. The polynucleotide of a transgene can be an exogenous polynucleotide. A fragment (e.g., a functional fragment) of a protein can comprise at least or at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the amino acid sequence of the protein. [91] As used herein, “transgenic organisms”, generally refers to recombinant organisms in which a desired DNA sequence or genetic locus within the genome of an organism is modified by insertion, deletion, substitution, or other manipulation of nucleotide sequences.

[92] As used herein, the term “transgenic plant” or “transgenic fungi: refers respectively to a plant or progeny of a plant, refers to a fungus or progeny fungus of any subsequent generation derived therefrom, wherein the DNA of the plant or progeny thereof, or fungus or progeny thereof contains an introduced exogenous DNA segment not naturally present in a non-transgenic plant of the same strain. The transgenic plant may additionally contain sequences which are native to the plant being transformed, but wherein the “exogenous” gene has been altered in order to alter the level or pattern of expression of the gene, for example, by use of one or more heterologous regulatory or other elements.

[93] As used herein, a gene sequence can be referred to as “unmasked” when the sequence does not include any linker or promoter sequences.

[94] As used herein, a “vector” or a “plasmid” refers to a polynucleotide (e.g., DNA or RNA), which can be used for and/ or can function as a vehicle to carry genetic material into a cell, where it can be replicated and/or expressed. In some embodiments, a vector is agrobacterium transformation vector. In some instances, the vector is a yeast artificial chromosome, phagemid, bacterial artificial chromosome, virus, or linear DNA (e.g., linear PCR product), for example, or any other type of construct useful for transferring a polynucleotide sequence into another cell. A vector (or portion thereof) can exist transiently (i.e., not integrated into the genome) or stably (i.e., integrated into the genome) in the target cell. In some embodiments, a vector can further comprise a selection marker or a reporter.

[95] The term “heteroaryl” and “heteroaryl ring” may be used interchangeably, and refers to an aromatic ring comprising at least one ring atom that is a heteroatom, such as O, N, or S.

[96] The term “heterocyclyl”, “heterocycle”, and “heterocyclic ring” may be used interchangeably, and refer to a non-aromatic hydrocarbon ring containing 3 to 12 atoms unless stated otherwise, comprising at least one ring atom that is a heteroatom, such as O, N, or S. “Heterocyclyl” rings encompass monocyclic, bicyclic, tricyclic, polycyclic, bridged, fused, and spiro rings, including mono spiro and di spiro rings.

[97] “ Substituted,” whether preceded by the term “optionally” or not, indicates that at least one hydrogen of the substituted group is replaced by a substituent.

[98] As used herein, a “genetically engineered cell” or “a genetically engineered organism” can be an organism comprising one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism’s genome. For example, genetic modification can refer to alterations, additions, and/or deletion of one or more genes. A genetically modified cell can also refer to a cell with an added, deleted and/or altered gene.

[99] References to a percentage sequence identity between two nucleotide sequences means that, when aligned, that percentage of nucleotides are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs know in the art, for example those described in section 7.7.18 of Current Protocols in Molecular Biology (F. M. Ausubel et al, eds., 1987) Supplement 30, which is incorporated by reference. A preferred alignment is determined by the Smith - Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith - Waterman homology search algorithm is disclosed in Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489, which is incorporated by reference.

[100] As used herein, “substantially pure” means sufficiently homogeneous to appear free of readily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those of skill in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of the compounds to produce substantially chemically pure compounds are known to those of skill in the art. A substantially chemically pure compound may, however, be a mixture of stereoisomers. In such instances, further purification might increase the specific activity of the compound. In some embodiments, the compositions of the present disclosure are substantially pure.

[101] As used herein, “wild type” or “wild-type organism” or “wild type species” refers to an organism that has a genotype or a phenotype of a typical organism of a species as it occurs in nature.

[102] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, geometric (or conformational) forms of the structure; for example, the R and S configurations for each asymmetric center, (Z) and (E) double bond isomers, and (Z) and (E) conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the disclosure.

[103] Although various features of the disclosure may be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the disclosure may be described herein in the context of separate embodiments for clarity, various aspects and embodiments can be implemented in a single embodiment.

OVERVIEW

Alkaloids

[104] Alkaloidal compounds represent one of the broadest classes of secondary metabolites in plant and fungal species. Some alkaloids have unprecedented therapeutic profiles (e.g., psilocybin) and possess dramatic antimicrobial, antitumor, and in some cases psychotropic profiles. One particular subclass of alkaloids gaining traction in the therapeutic space are psychotropic alkaloids. These compounds, including psilocybin, are derived from the classical L- tryptophan amino acid (4), possessing a central indole bicyclic ring system. While most therapeutic research, to date, focuses on psilocybin and its dephosphorylated active metabolite, psilocin (2), there are other derivatives and analogues of these compounds with dramatic therapeutic potential for applications in treating mental illness and disorders. While psilocybin (1) and psilocin (2) are known to be produced in trace amounts in fungal species, the related compounds produced in the biosynthetic pathway can be even more difficult to quantify, as some are produced in trace amounts, and some are not produced in detectable amounts using conventional diagnostic and analytical methods.

Psilocybin (1) Psilocin (2) N,N-dimethyltryptamine (3) Tryptamine (4) L-tryptophan (5)

[105] Few compound scaffolds have demonstrated psychotropic effects like psilocybin. This can be attributed to its ability to cross the blood-brain barrier. While some alkaloids are simple building blocks for more complex structures, alkaloids like the amino acid, L-tryptophan (5), can be manipulated through diverse biosynthetic pathways to produce unique, and medicinally potent drug products. In some embodiments, a derivative, analog, or metabolite of L-tryptophan (5) is produced.

[106] In some embodiments, are the increased production of alkaloids found in the psilocybin biosynthetic pathway such as N,N-dimethyltryptamine (3) and tryptamine (4).

[107] In some embodiments, the alkaloid produced is new-to-nature, meaning it is not naturally occurring in comparable organisms without any genetic modification.

[108] In some embodiments the alkaloid produced by the genetically modified fungal cell includes compounds of Formula (I). [109] Formula (I) encompasses compounds falling within the following structure:

and includes tautomers of those compounds and pharmaceutically acceptable salts of any of the foregoing wherein:

-R¹ is H or -CH₃;

-R² is H, -OH, -OP(O)(OM)₂, -C(O)CH₃, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

-R³ is H, -OH, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

-R⁴ is H, -OH, or -OCH₃;

-R⁵ is H, -OH, -OP(O)(OM)₂, or -OCH₃;

-R^A is H, -COOH;

-R^x, R^y, and R^z are each independently H, -CH₃, -C(O)CH₃, -CH₂CH₃, or -CH(CH₃)₂; and -M is H, Li⁺, Na⁺, K⁺ or is absent.

[HO] In some embodiments the alkaloid produced by the genetically modified fungal cell includes compounds of Formula (la).

[Hl] Formula (I) includes compounds of Formula (la). Formula (la) encompasses compounds falling within the following structure:

and includes tautomers of those compounds and pharmaceutically acceptable salts of any of the foregoing wherein: -R¹ is H or -CH₃;

-R³ is H, -OH, -OCH3, -OCH2CH3, or -OCH(CH₃)2;

-R⁴ is H, -OH, or -OCH3;

-R⁵ is H, -OH, -OP(O)(OM)₂, or -OCH3;

-R^A is H, -COOH;

-R^x, R^y, and R^z are each independently H, -CH3, -C(O)CH3, -CH2CH3, or -CH(CH3)2; and -M is H, Li⁺, Na⁺, K⁺ or is absent.

[112] Formula (I) includes compounds of Formula (lb). Formula (lb) encompasses compounds falling within the following structure:

-R¹ is H or -CH3;

-R³ is H, -OH, -OCH3, -OCH2CH3, or -OCH(CH₃)2;

-R⁴ is H, -OH, or -OCH3;

-R⁵ is H, -OH, -OP(O)(OM)₂, or -OCH3;

-R^A is H, -COOH;

[113] In some embodiments the alkaloid produced by the genetically modified fungal cell includes compounds of Formula (Ic).

[114] Formula (I) includes compounds of Formula (Ic). Formula (Ic) encompasses compounds falling within the following structure:

-R¹ is H or -CH₃;

-R³ is H, -OH, -OCH3, -OCH2CH3, or -OCH(CH₃)2;

-R⁴ is H, -OH, or -OCH3;

-R⁵ is H, -OH, -OP(O)(OM)₂, or -OCH3;

-R^A is H, -COOH;

[115] In some embodiments, a compound of Formula (I) can be an alkaloid selected from:

Psilocybin (1), Psilocin (2), N,N-dimethyltryptamine (3), Tryptamine (4), L-tryptophan (5),

deph baeocystin (9), norbaeocystin (10),

Norpsilocin (6), aeruginascin (7), osphorylated aeruginascin (8),

4-hydroxy-tryptophan (15), 4-hydroxy-tryptamine ( 16), N-acetyl-4-hydroxytryptamine (17),

5-methoxy-N,N-diisopropyltryptamine (18), and 4-acetoxy-N,N-dimethyltryptamine (19).

[116] In some embodiments, the alkaloid produced is a derivative or analogue of any one of compounds 1-19. In some embodiments the alkaloid produced comprises an N-methylated derivative of any one of compounds 1-19. In some embodiments, the embodiments the alkaloid produced is an N-ethylated derivative of any one of compounds 1-19. In some embodiments, the embodiments the alkaloid produced is an N-propylated derivative of any one of compounds 1-19. In some embodiments, the genetically modified fungus produces at least two of compounds 1-19. In some embodiments, an extract of the genetically modified fungus contains at least one of compounds 1-19. In some embodiments, the alkaloid is an analogue of psilocybin wherein the analogue contains a 4-acetoxy group (e.g., 4-acetoxy-N,N-dimethyltryptamine). In some embodiments, the alkaloid comprises 4-acetoxy-N,N-dimethyltryptamine.

[117] Another class of alkaloids also derived from L-tryptophan (4), are the harmala alkaloids (e.g., harmane, and harmine) which contain a P-carboline core scaffold. Harmala alkaloids have been detected in P. cubensis fungi at very low concentrations around 0.2 pg/g. Harmala alkaloids are neuroactive compounds that inhibit monoamine oxidase (MAO) which degrades psilocybin in the body. In some instances, a monoamine oxidase can be: an MAO A, an MAO B, or a combination of MAO A and MAO B. In some instances, an inhibitor of MAO A, MAO B, or MAO can be: i) contain any composition or pharmaceutical composition herein; or ii) administered concurrently or consecutively by a same or different route of administration along with a composition or a pharmaceutical composition herein. Thus, the presence of P-carboline containing alkaloids cam contribute to the prevention of psilocybin degradation in the human body. Exemplary P-carboline containing alkaloids include harmine, harmaline, harmalol, tetrahydroharmine, harmaline, isoharmine, harmine acid methyl ester, harminilic acid, harmanamide, and acetylnorharmine, and derivatives and analogues thereof.

[118] In some embodiments the alkaloid produced by the genetically modified fungal cell includes compounds of Formula (II).

[119] Formula (II) encompasses compounds falling within the following structure:

-X is N, or NH;

- Y is -C-, -CH-, or -CH₂-;

-Ring A is a 5-membered heterocyclyl or heteroaryl;

-Ring B is a 6-membered heterocyclyl or heteroaryl;

-R¹ is H or -CH₃;

-R² and R³ are each independently selected from H, -OH, and -OCH₃;

-R^Ais H, -CH₃, -C(O)CH₃, -C(O)OCH₃, -C(O)NH₂, -C(O)OH; and

-R^B is absent, H, -CH₃, or -COOH. [120] In some embodiments the alkaloid produced by the genetically modified fungal cell includes compounds of Formula (Ila).

[121] Formula (II) includes compounds of Formula (Ila). Formula (Ila) encompasses compounds falling within the following structure:

[122] Formula (Ila) encompasses compounds falling within the following structure:

-R² and R³ are each independently selected from H, -OH, and -OCH₃; and

-R^Ais H, -CH₃, -C(O)CH₃, -C(O)OCH₃, -C(O)NH₂, -C(O)OH;

[123] In some embodiments the alkaloid produced by the genetically modified fungal cell includes compounds of Formula (lib).

[124] Formula (II) includes compounds of Formula (lib). Formula (lib) encompasses compounds falling within the following structure:

[125] Formula (lib) encompasses compounds falling within the following structure:

-R² and R³ are each independently selected from H, -OH, and -OCH₃;

-R^Ais H, -CH₃, -C(O)CH₃, -C(O)OCH₃, -C(O)NH₂, -C(O)OH; and

-R^B is H, -CH₃, or -COOH.

[126] In some embodiments, a compound of Formula (II) can be an alkaloid selected from:

harmalane (34), isoharmine (35), Methyl 7-methoxy-beta- methyl 7-methoxy-1 -methyl-2, 3,4,9- carboline-1 -carboxylate (36), tetrahydro-1 /-/-pyrido[3,4-b]indole-1 - carboxylate (37),

harmanilic acid (38), harmanamide (39), and acetylnorharmine (40).

[127] In some embodiments, the alkaloid produced is a derivative or analogue of any one of compounds 30-40. In some embodiments the alkaloid produced comprises an N-methylated derivative of any one of compounds 30-40. In some embodiments, the embodiments the alkaloid produced is an N-ethylated derivative of any one of compounds 30-40. In some embodiments, the embodiments the alkaloid produced is an N-propylated derivative of any one of compounds 30- 40. In some embodiments, the genetically modified fungus produces at least two of compounds 30-40. In some embodiments, the genetically modified fungus or an extract thereof comprises at least one compound selected from compounds 1-19 and further comprises at least one compound selected from compounds 30-40.

[128] In some cases, the genetic modifications described herein allow for the production of alkaloids at increased amounts as measured by % dry weight as compared to that of a comparable unmodified organism (i.e., a wild-type organism). In some cases, the alkaloid is a secondary metabolite. In some cases, the alkaloid is tryptophan derived alkaloid. For example, the alkaloid can be psilocybin or a derivative or analog thereof. In some cases, the alkaloid is psilocin. In some cases, the alkaloid can be baeocystin. In some cases, the alkaloid can be norbaeocystin. In some cases, the alkaloid can be dimethyltryptamine. In some cases, the alkaloid can be tryptamine. In some cases, the alkaloid can be 4-hydroxytryptamine. In some cases, the alkaloid can be N,N- dimethyltryptamine. In some cases, the alkaloid can be serotonin. In some cases, the alkaloid can be melatonin. In some cases, the alkaloid can be melanin. In some cases, the alkaloid can be N- acetyl-hydroxytryptamine. In some cases, the alkaloid can be 4-hydroxy-L-tryptophan. In some cases, the alkaloid can be 5-hydroxy-Z-tryptophan. In some cases, the alkaloid can be 7-hydroxy- Z-tryptophan. In some cases, the alkaloid can be 4-phosporyloxy-N,N-dimethyltryptamine. In some cases, the alkaloid can be aeruginascin. In some cases, the alkaloid can be isonorbaeocystin. In some cases, the alkaloid can be 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l- aminium, 4-phosphoryloxy-N,N-dimethytryptamine, or P-carboline. In some cases, the alkaloid can be ketamine. In some cases, the alkaloid can be normelatonin. In some cases, the alkaloid can be 3,4-methylenedioxymethamphetamine. In some cases, the alkaloid can be P-carboline.

[129] In some cases, a combination of alkaloids can be produced at higher concentrations measured by % dry weight. In some cases, the alkaloids include any one or more of psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, baeocystin, norbaeocystin, isonorbaeocystin, serotonin, melatonin, melanin, N-acetyl-hydroxytryptamine, 4-hydroxy-L- tryptophan, 5 -hydroxy -L-tryptophan, 7-hydroxy-L-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan-l-aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, normelatonin, 3, 4-methylenedi oxymethamphetamine, and any derivative and any analogue thereof.

[130] In some embodiments, the genetically modified fungus further comprises a non-naturally occurring alkaloid. In some embodiments, the genetically modified fungus comprises a non- naturally occurring harmala alkaloid. In some embodiments, the genetically modified fungus comprises N, N-dimethyltryptamine and a harmala alkaloid.

Production of alkaloids in genetically modified organisms

[131] In some embodiments, the genetic modifications described herein allow for the production of alkaloids at increased amounts as measured by % dry weight as compared to that of a comparable unmodified organism (i.e., a wild-type organism). In some cases, the alkaloid is a secondary metabolite. In some embodiments, the genetically modified fungus further comprises a non-naturally occurring alkaloid. In some embodiments, the genetically modified fungus comprises- a non-naturally occurring harmala alkaloid. In some embodiments, the genetically modified fungus comprises N, N-dimethyltryptamine and a harmala alkaloid. In some cases, the alkaloid is a neuroactive alkaloid. In some cases, the alkaloid is a psychotropic alkaloid. In some cases, the alkaloid is a neuroactive alkaloid. In some cases, the alkaloid is a psychotropic alkaloid. In some cases, the alkaloid is a tryptophan-derived alkaloid. For example, the alkaloid can be psilocybin or a derivative or analog thereof. In some cases, the alkaloid is psilocin. In some cases, the alkaloid can be baeocystin. In some cases, the alkaloid can be tryptamine. In some cases, the alkaloid can be 4-hydroxytryptamine. In some cases, the alkaloid can be N, N-dimethyltryptamine. In some cases, the alkaloid can be serotonin. In some cases, the alkaloid can be melatonin. In some cases, the alkaloid can be melanin. In some cases, the alkaloid can be N-acetyl-hydroxytryptamine. In some cases, the alkaloid can be 4-hydroxy-L- tryptophan. In some cases, the alkaloid can be 5-hydroxy-L-tryptophan. In some cases, the alkaloid can be 7-hydroxy-L-tryptophan. In some cases, the alkaloid can be 4-phosporyloxy- N,N-dimethyltryptamine. In some cases, the alkaloid can be aeruginascin. In some cases, the alkaloid can be 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium, 4- phosphoryloxy-N,N-dimethytryptamine. In some cases, the alkaloid can be ketamine. In some cases, the alkaloid can be normelatonin. In some cases, the alkaloid can be 3,4- methylenedioxymethamphetamine. In some cases, the alkaloid can be a P-carboline.

[132] In some embodiments, a combination of alkaloids can be produced at higher concentrations measured by % dry weight. In some cases, the alkaloids include any one or more of psilocybin, norpsilocin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, baeocystin, norbaeocystin, serotonin, melatonin, melanin, N-acetyl-hydroxytryptamine, 4- hydroxy-L-tryptophan, 5-hydroxy-L-tryptophan, 7-hydroxy-L-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan-l-aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, normelatonin, 3, 4-methylenedi oxymethamphetamine, P-carboline, or any derivative or any analogue thereof.

[133] For example, in some cases, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of a tryptamine-derivative alkaloid or a tryptophan-derivative alkaloid as measured by dry weight of and as compared to a comparable control without genetic modification.

[134] In some cases, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of dimethyltryptamine, or a derivative thereof, as measured by dry weight and as compared to a comparable control without genetic modification.

[135] In some cases, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of psilocybin, or a derivative thereof, as measured by dry weight and as compared to a comparable control without genetic modification.

[136] In some cases, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of psilocin as measured by dry weight and as compared to a comparable control without genetic modification.

[137] In some cases, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of norpsilocin as measured by dry weight and as compared to a comparable control without genetic modification.

[138] In some cases, the genetically modified organism can comprises about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of norbaeocystin or baeocystin, or a derivative thereof, as measured by dry weight of and as compared to a comparable control without genetic modification.

[139] In some cases, the genetically modified organism can comprise about 10% 20%, 25%,

30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of serotonin, melatonin, melanin, N,N-dimethytryptamine, N-acetyl- hydroxytryptamine, serotonin, aeruginascin, isonorbaeocystin, 2-(4-Hydroxy-lH-indol-3-yl)- N,N,N-trimethylethan-l-aminium, ketamine, normelatonin, 3,4- methylenedioxymethamphetamine, N, N-Dimethyltryptamine, or any derivative thereof, as measured by dry weight and as compared to a comparable control without genetic modification. In some cases, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of psilocybin, psilocin, aeruginascin, Baeocystin, norbaeocystin, norpsilocin, melatonin, melanin, N-acetyl-hydroxytryptamine, serotonin, aeruginascin, isonorbaeocystin, 2-(4-Hydroxy-lH-indol- 3-yl)-N,N,N-trimethylethan-l-aminium, ketamine, normelatonin, 3,4- methylenedioxymethamphetamine, N, N-Dimethyltryptamine, or any derivative thereof, as measured by dry weight and as compared to a comparable control without genetic modification.

[140] In some cases, the detection of tryptamine derivatives, or secondary metabolites produced by the genetically modified organism can be characterized by LCMS. In some cases, the detection of tryptamine derivatives, or secondary metabolites produced by the genetically modified organism can be characterized by MALDI-TOF. In some cases, the detection of tryptamine derivatives, or secondary metabolites produced by the genetically modified organism, can be characterized by any comparable analytical technique to LCMS or GCMS.

[141] In some embodiments, the genetically modified organism is analyzed using conventional methods to identify and/or quantify the amount of a secondary metabolite described herein, present in the genetically modified organism.

[142] In some embodiments, the genetically modified organism produces an elevated amount of N,N, -dimethyltryptamine in comparison to a naturally occurring otherwise equivalent genetically modified organism. In some embodiments the genetically modified organism expresses a gene product as shown in TABLE 4A or TABLE 4B. In some embodiments the genetically modified organism expresses a gene product with 95% sequence identity to any one of SEQ ID NOs: 17-28, and 229-230. In some embodiments the genetically modified organism expresses a gene product that comprises any one of SEQ ID NOs: 17-28, and 229-230. In some embodiments the genetically modified organism expresses a gene product that is any one of SEQ ID NOs: 17-28, and 229-230.

Production of Harmala Alkaloids

[143] Another class of alkaloids also derived from L-tryptophan (4), are the harmala alkaloids (e.g., harmane, and harmine) which contain a P-carboline core scaffold. Harmala alkaloids have been detected in P. cubensis fungi at very low concentrations around 0.2 pg/g. Harmala alkaloids are neuroactive compounds that inhibit monoamine oxidase (MAO) which degrades psilocybin in the body. Thus, the presence of monoamine oxidase inhibitor, which can be a P-carboline- containing alkaloid can contribute to the prevention of psilocybin, or DMT degradation (that is, increased the half-life of psilocybin or DMT) in the human body. Thus, the presence of P- carboline-containing alkaloids can contribute to the prevention of DMT degradation in the human body. In some embodiments, inhibition of a PsiH gene can result in an increased production of a harmala alkaloid described herein. Exemplary P-carboline containing alkaloids include harmine, harmaline, harmalol, tetrahydroharmine, harmaline, isoharmine, harmine acid methyl ester, harminilic acid, harmanamide, and acetylnorharmine, and derivatives and analogues thereof. In some embodiments, a harmala alkaloid is produced by a genetically modified organism described herein. In some embodiments the harmala alkaloid can be one of the following:

harmanilic acid (38), harmanamide (39), and acetylnorharmine (40).

[144] In some embodiments, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound harmine, or a derivative thereof, as measured by dry weight of and as compared to a comparable control without genetic modification.

[145] In some embodiments, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of harmaline, or a derivative thereof, as measured by dry weight of and as compared to a comparable control without genetic modification.

[146] In some embodiments, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of harmalol, or a derivative thereof, as measured by dry weight of and as compared to a comparable control without genetic modification.

[147] In some embodiments, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of 1,2,3,4-tetrahydroharmine, or a derivative thereof, as measured by dry weight of and as compared to a comparable control without genetic modification.

[148] In some embodiments, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of harmalane, or a derivative thereof, as measured by dry weight of and as compared to a comparable control without genetic modification.

[149] In some embodiments, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of isoharmine, or a derivative thereof, as measured by dry weight of and as compared to a comparable control without genetic modification.

[150] In some embodiments, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of methyl-7-methoxy-beta-carboline-l-carboxylate, or a derivative thereof, as measured by dry weight of and as compared to a comparable control without genetic modification.

[151] In some embodiments, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of methyl-7-methoxy-methyl-2,3,4,9-tetrahydro-lH-pyrido[3,4 P]- indole-1 -carboxylate, or a derivative thereof, as measured by dry weight of and as compared to a comparable control without genetic modification.

[152] In some embodiments, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of harmanilic acid, or a derivative thereof, as measured by dry weight of and as compared to a comparable control without genetic modification.

[153] In some embodiments, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of harmanamide, or a derivative thereof, as measured by dry weight of and as compared to a comparable control without genetic modification.

[154] In some embodiments, the genetically modified organism can comprise about 10% 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, 150%, 175%, 200%, and up to 400% percent more of a compound of acetylnorharmine, or a derivative thereof, as measured by dry weight of and as compared to a comparable control without genetic modification. [155] In some embodiments, the detection of tryptamine derivatives, or secondary metabolites produced by the genetically modified organism can be characterized by LCMS. In some cases, the detection of tryptamine derivatives, or secondary metabolites produced by the genetically modified organism can be characterized by MALDI-TOF. In some cases, the detection of tryptamine derivatives, or secondary metabolites produced by the genetically modified organism, can be characterized by any comparable analytical technique to LCMS or GCMS.

[156] In some embodiments, the genetically modified organism is analysed using conventional methods to identify and/or quantify the amount of a secondary metabolite described herein, present in the genetically modified organism. In some embodiments, a genetically engineered fungus comprises harmala alkaloids (e.g., harmane, and harmine). In some embodiments, the amount of a harmala in a genetically engineered fungus described herein has an increased amount of the harmala alkaloid in comparison to a comparable wild type fungus.

Analysis of alkaloids produced in genetically modified organisms

[157] In some embodiments, the alkaloids are quantified by LC-MS. In some embodiments, samples for quantification are first freeze dried and then quantified by dry weight analysis. In some embodiments, the free-dried samples are quantified for alkaloids by LC-MS. In some embodiments, the alkaloids are quantified by HRMS. In some embodiments, the alkaloids are detected by HRMS. In some embodiments, the alkaloids are further extracted from the fungal tissue samples and then analyzed. In some embodiments, the alkaloids are analyzed by ’H NMR, ¹³C, or ³¹P NMR. In some embodiments, the alkaloids are analyzed by ^XH NMR, ¹³C, and ³¹P NMR. In some embodiments, the alkaloids are purified prior to analysis. In some embodiments the alkaloids are isolated and purified. In some embodiments the alkaloids are isolated and purified by a chromatographic method. In some embodiments the alkaloids are isolated and purified using HPLC. In some embodiments the alkaloids are isolated and purified using UPLC or UHPLC. In some embodiments, the alkaloids are not isolated or purified and are analyzed in the fungal sample, directly. In some embodiments an extract of the alkaloid or alkaloids isolated from a genetically modified fungus are prepared. In some embodiments, the alkaloids may be purified and isolated, separately. In some embodiments, the alkaloidal content is measured as aggregate alkaloidal content meaning the amount includes the net alkaloidal content of multiple alkaloid compounds produced by a genetically modified fungal cell.

[158] In some embodiments, the alkaloid produced results from a genetic modification to a gene within the psilocybin biosynthetic pathway. In some embodiments, the alkaloid produced results from a genetic modification to a gene near the psilocybin biosynthetic pathway gene cluster.

Genetically modified organisms [159] In some embodiments, a genetically modified organism provided herein is a multicellular organism. In some embodiments, a genetically modified organism is a unicellular organism, embodiments, the genetically modified organism is a single plant cell or a single fungal cell. Embodiments described herein also include populations of cells, for instance a population of cells from a fungal species. For example, in some embodiments, the genetically modified organism comprises a population of genetically modified fungal cells that collectively form a mycelial mass. In some embodiments, a genetically modified organism as provided herein is from a fungus. For example, in some cases, the genetically modified organism provided herein is a fungal cell. In some cases, the fungus or fungal cell is from the division Basidiomycota. In some cases, the Basidiomycota fungus or fungal cell can be from Psilocybe, Conocybe, Gymnopilus, Panaeolus, Pluteus, or Stropharia. In some embodiments, the fungus or fungal cell is from Gymnopilus dilepis. In some cases, the fungus or fungal cell is from Pluteus salicinus. In some cases, the fungus or fungal cell is from Psilocybe cubensis. In some cases, the fungus or fungal cell is from Panaeolus cyanescecens. In some cases, the fungus or fungal cell is from Pleurotus nebrodensis. In some cases, the fungal cell is a mycelium, or mycelial cell. In some embodiments, the fungal cell is an aerial mycelium. In some cases, the fungal cell is a fungal protoplast. In some embodiments, the protoplast is isolated from a mycelium, or mycelial mass. In some cases, a mycelial mass is present and comprises the fungal cells.

[160] In some embodiments, a genetically modified organism described herein is a plant. For example, in some embodiments, the genetically modified organism is from the genus Cannabis. In some cases, a genetically modified organism described herein is a bacterium. In some cases, a bacterium is an agrobacterium.

[161] In some embodiments, a genetically modified organism described herein comprises Mitragyna speciosa (commonly known as kratom). Kratom is a tropical evergreen tree in part of the coffee family, which is native to Southeast Asia. Kratom is indigenous to Thailand, Indonesia, Malaysia, Myanmar, and Papua New Guinea, where it has been used in herbal medicine since at least the nineteenth century. Kratom has opioid properties and some stimulant-like effects. In some embodiments, compositions and methods described herein are used to produce a genetically modified kratom having increased opioid or stimulate-like properties.

[162] In some embodiments, a genetically modified organism described herein is a plant. In some cases, a genetically modified organism described herein is a bacterium. In some cases, a bacterium is an agrobacterium. In some embodiments, a genetically modified organism can be a eukaryotic organism. In some embodiments, a genetically modified organism described herein can be a fungus. In some embodiments, a genetically modified organism can be of the phylum basidiomycota. In some embodiments, a genetically modified organism can be from a genera selected from Copelandia, Gymnopilus, Inocybe, Panaeolus, Pholiotina, Pluteus, and Psilocybe.

[163] In some embodiments, a genetically modified organism described herein can be a multicellular organism. In some instances, a genetically modified organism can be a unicellular organism. For example, in certain embodiments, the organism can be a single plant cell or a single fungal cell. Embodiments described herein also include populations of cells, for instance a population of cells from a fungal species described herein. For example, in some embodiments, the genetically modified organism comprises a population of genetically modified fungal cells that collective form a mycelial mass. In some embodiments, the fungal cell comprises mycelium.

[164] In some embodiments, this disclosure provides genetically modified organisms that are genetically modified to promote the expression of tryptamine monooxygenases. For example, the genetically modified organisms can comprise a genetic modification that results in an increased expression of tryptamine monooxygenases as compared to a comparable wild-type organism. One tryptamine monooxygenase of interest is that produced by PsiH2. A phylogenic analysis of PsiH2 reveals a high degree of sequence diversity suggesting the gene has changed over time during evolution to improve finesses of certain fungal species and thereby adapt. Thus, the heterologous introduction of the PsiH2 gene from one fungal species to another can alter the gene product expression, and in turn influence the identity and quantity of alkaloids produced by the modified fungal organism. Accordingly, the genetically modified organism as described herein can be useful to make an increased amount of a tryptophan-derived alkaloid (e.g., psilocybin) as compared to a comparable wild-type organism.

[165] In some embodiments, this disclosure provides a genetically modified organism that is genetically modified to promote production of psilocybin. For example, in some embodiments this disclosure provides a genetically modified organism that is modified to reduce or eliminate expression of an alkaline phosphatase, e.g., psilocybin phosphatase, which can dephosphorylate psilocybin thereby converting psilocybin into psilocin. By suppressing or eliminating the activity of the alkaline phosphatase, the genetically modified organism can comprise a higher concentration of psilocybin as compared to a comparable wild-type organism, i.e., a comparable organism without the genetic modification. In some embodiments, the additional modulation of PsiR, can influence the production of alkaloid synthesis.

[166] In some embodiments, PsiR is introduced as an exogenous nucleotide. In different species fungal species, the order of the psilocybin producing gene cluster contains discrepancies with respect to its transcriptional regulators (e.g., PsiR). The diversity in composition can suggest that there are alternative routes of psilocybin production, and/or additional biosynthetic pathways capable of producing non-naturally occurring alkaloids beyond the psilocybin scaffold. It is known that all psilocybin producing mushrooms contain a transcriptional regulator, PsiR, though its placement varies, as discussed above. PsiR is a basic Helix-Loop-Helix (bHLH) transcriptional regulator expressed in fruiting bodies. BHLH are known to bind DNA to a consensus hexanucleotide sequence known as the E-box (CANNTG (SEQ ID NO: 150)). Other genes in the psilocybin biosynthesis gene cluster which also contain one E-box motif in their promoters are PsiD, PsiH, PsiM, and PsiT2. PsiTl has two E-box motif regions. Interestingly, PsiP contains 4 E- box motifs (500 base pairs upstream of ATG). PsiL and PsiK do not have this promoter region. When a fungus includes multiple PsiP genes, the genes or their protein expression products referenced herein may be numbered to differentiate, e.g., PsiPl and PsiP2.

[167] In some embodiments, the genetically modified organism can comprise one or more genetic modifications. For example, in some embodiments, the genetically modified organism can comprise a genetic modification that results in an increased expression of a tryptamine monooxygenase as compared to a comparable wild-type organism (e.g., PsiH, and PsiH2), and a genetic modification that modulates transcriptional regulation function, e.g., psilocybin phosphatase. In different embodiments, the genetically modified organism can comprise a genetic modification that results in increased expression of a tryptamine monooxygenase as compared to a comparable wild-type organism, and a genetic modification that results in increase expression of a heterologous transcription factor. In some embodiments, the genetically modified organism can comprise a genetic modification that results in modulation of a psilocybin biosynthesis enzyme. For example, in some embodiments, the genetically modified organism can comprise a genetic modification that results in an increased expression of L-tryptophan decarboxylase as compared to a comparable wild-type organism, and a genetic modification that results in decreased expression or activity of an alkaline phosphatase, e.g., psilocybin phosphatase. In different embodiments, the genetically modified organism can comprise a genetic modification that results in increased expression of L-tryptophan decarboxylase as compared to a comparable wild-type organism, and a genetic modification that results in increase expression of a 4-hydroxytryptamine kinase as compared to a comparable wild-type organism. In some embodiments, the genetically modified organism can comprise a genetic modification that results in increased expression of L- tryptophan decarboxylase as compared to a comparable wild-type organism, a genetic modification that results in increased expression of a 4-hydroxytryptamine kinase as compared to a comparable wild-type organism, and a genetic modification that results in reduced expression of psilocybin phosphatase as compared to a comparable wild-type organism. Additionally, in other embodiments, the genetically modified organism can further comprise a genetic modification that results in increased expression of a methyltransferase, such as the methyltransferase encoded by PsiM. The genetically modified organism can also further comprise a genetic modification that results in increased expression of a P450 monoxygenase as compared to a comparable wild-type organism.

[168] Accordingly, genetically modified organisms described herein can include one or more genetic modifications that result in any one of (a) increased tryptophan decarboxylation, (b) increased tryptamine 4-hydroxylation, (c) increased 4-hydroxytryptamine O-phosphorylation, (d) increased psilocybin via sequential A-methylations, or (e) reduced expression of a psilocybin phosphatase as compared to a control organism without the genetic modification. The genetically modified organism can further include any one or more of genetically modifications described in WO 2021/067626, which is incorporated by reference.

[169] For example, in some embodiments the genetically modified organism includes a genetic modification that results in (i) upregulated expression of a tryptophan decarboxylase gene, a psilocybin-related hydroxylase gene, a psilocybin-related N-m ethyltransferase gene, or a psilocybin-related phosphotransferase gene; (ii) synthesis of non-psilocybin alkaloids; (iii) increased production of tryptophan in the genetically modified organism compared to a comparable control organism without the genetic modification; (iv) production of novel alkaloids; and (v) increased production of alkaloids detected in non-genetically modified organisms. As a result of the genetic modification the genetically modified organism can produce an increased amount of a compound, such as, for example, compounds selected from:

Tryptamine, DMT, Psilocin, Psilocybin as compared to production of the same compound in a comparable control organism without the genetic modification.

[170] In some embodiments, compositions, systems, and methods of this disclosure can produce a genetically modified organism including a genetic modification that results in the genetically modified organism exhibiting a phenotype that is visually distinct from a phenotype of a comparable wild-type organism. For example, in some embodiments, the phenotype comprises a blue coloration. The phenotype can be measured using methods known in the art, for example, the phenotype comprising the blue coloration can be measured using a spectrophotometer. The spectral reflectance, of the genetically modified organism, in the wavelength region from 400 to 525 nm (the blue regions) can be high, and the spectral reflectance for wavelengths longer than 550 nm can be low. Conversely, a comparable wild-type organism can be described as have a spectral reflectance in the wavelength region from 400 nm to 525 nm that is substantially lower than the genetically modified organism. In some embodiments, the genetically modified organism is a mycelial mass comprising the blue phenotype. In some embodiments, a phenotypic distinction can include a change in color of a fungus, or portion thereof, from a color of the fungus, or portion thereof, prior to a genetic change or modification of the fungus, or portion thereof. In some embodiments, a phenotypic distinction can include a change in color, shape, length, mass, thickness, density, or any combination of these, of a fungus, or portion thereof, from a color, shape, length, mass, thickness, density, of the fungus, or portion thereof, prior to a genetic change or modification of the fungus, or portion thereof. In some embodiments, a fungus can include or be a mature fungus, a fruiting body, a mycelial mass, primordial cells, or any combination of these. In some embodiments, the genetically modified organism comprises a fungus with the blue phenotype. In other embodiments, a portion of the genetically modified organism comprises the blue phenotype, for example, an inner portion of tissue upon exposure to air. Because of the association between the blue phenotype and increased alkaloid content, in some embodiments, the blue phenotype is used as a reporter of alkaloidal content, e.g., psilocin.

[171] In some embodiments, this disclosure involves an increased expression of L-tryptophan decarboxylase in a fungus or fungal cell can alter a phenotype of the fungus or fungal cell. In some embodiments, this disclosure involves methods of assessing whether a fungal organism is genetically modified or assessing to what extent a fungal organism expresses an alkaloid such as psilocybin or psilocin based on a blue coloration of the organism.

[172] In some embodiments, the genetically modified organism can include a genetic modification. In some embodiment the genetic modification is to a gene that results in the upregulation or down regulation of a gene product in the genetically modified organism. For example, the gene can be a gene described in TABLES 1, 2, 3A, and 3B or that comprise a sequence that is at least, for example, 65%, 75%, 85%, 90%, 95%, 99%, or 100% identical to one of the sequences listed in TABLES 1, 2, 3A, and 3B. In some embodiments, the gene comprises a sequence that is at least, for example, 65%, 75%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 1-14, 118-129, or 322. In some instances, the genetically modified organism includes a genetic modification that results in an increased expression of a gene product, for example, one or more of the gene products identified in TABLE 4A or TABLE 4B.

[173] TABLE 1 shows exemplary genes that can be upregulated or downregulated in a genetically modified organism. Length and number of introns of psilocybin biosynthetic genes for P. cubensis and P. cyanescens are shown. If there are two values in a cell, the first value refers to the respective gene of P. cubensis, the second to P. cyanescens. Values for P. cyanescens genes for PsiR, PsiTl, and PsiT2 of P. cubensis are predicted using the Augustus algorithm. An exemplary intron sequence is: gtttgtctctcgcttgcataccacccagcagctcactgatgtcgacttgtag (SEQ ID NO.: 672)

[174] In embodiments described herein, the genetically modified organism can include a genetic modification that results in the upregulation or down regulation of a gene. For example, a gene encoded by any one of the genes that are described in TABLE 1, or that comprise a sequence that is at least, for example, 65%, 75%, 85%, 90%, 95%, 99%, or 100% identical to one of the sequences listed in TABLE 2. In some embodiments, the gene comprises a sequence that is at least, for example, 65%, 75%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 1-14, 118-129, or 322.

[175] In some embodiments, the genetically modified organism includes a genetic modification that results in the upregulation or downregulation of a gene.

TABLE 1. Exemplary genes encoding gene products that can be upregulated or downregulated in genetically modified organisms.

Table 2. Exemplary genes and gene sequences encoding gene products that can be upregulated or downregulated in a genetically modified organism described herein.

Table 3A. P. cyanescence (Pcy) vector design for PsiH2 overexpression in P. cubensis

Table 3B. P. tampanensis (Pt) vector design for PsiH2 overexpression in P. cubensis

Table 4A. Exemplary PsiH and PsiH2 Amino Acid Sequences

TABLE 4B. Exemplary polypeptides that can be upregulated or downregulated in a genetically modified organism.

Methods of making genetic modifications

[176] Provided herein are methods for genetically modifying an organism, as described herein, by introducing an exogenous nucleic acid, e.g., a donor sequence, into a cell of the organism. Exemplary cells of the organisms include a fungal cell. Exemplary fungal cells include a protoplast. The exogenous nucleic acid may encode one or more gene products that, when expressed by the genetically modified organism, result in the genetically modified organism producing an increased amount of the one or more alkaloids as compared to a comparable wildtype organism. In some instances, the one or more genes can be one of the genes listed in TABLE 1 or TABLE 2. In some instances, one or more copies of the one or more genes included in TABLE 1 or TABLE 2 are provided by the exogenous nucleic acid. For example, in some instances at least 1, 2, 3, 4, 5, 6, or 7 copies of the one or more genes are introduced into the genetically modified organism with the exogenous nucleic acid. In some embodiments, the genetic modification results in a gene product described in TABLE 4A-TABLE 4B. In some embodiments, the genetically modified organism expressing one or more of the polynucleotides listed in TABLE 2, TABLE 3A-3B. In some cases, at least a portion of the exogenous nucleic acid can be integrated into the genome of the organism. For example, the exogenous nucleic acid can be inserted into a genomic break. In some instances, at least a portion of the exogenous nucleic acid includes sequences that are homologous to sequences flanking a target sequence for targeted integration. Methods of introducing an exogenous nucleic acid into a cell of an organism are generally known to the skilled artisan but may include the use of homology arms. In other instances, the exogenous nucleic acid can be randomly inserted into a genome of a target organism.

[177] In some embodiments, an exogenous nucleic acid can be integrated to the genome of the genetically modified organism by virtue of homologous recombination. Homologous recombination permits site specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the genome of the genetically modified organism.

[178] In some embodiments, the exogenous nucleic acid includes a promoter sequence. Increasing expression of designed gene products may be achieved by synthetically increasing expression by modulating promoter regions or inserting stronger promoters upstream of desired gene sequences. In some embodiments, for example, a gene promoter such as 35S gene promoter is used.

[179] In some embodiments, the exogenous nucleic acid can include a barcode or watermark sequence, which may be referred to as “a barcode”. A barcode can comprise a non-natural sequence. In some embodiments, the barcode can be used to identify transgenic organisms via genotyping. In some embodiments, the exogenous nucleic acid can include a selectable marker, such as an antibiotic resistance gene. Selectable marker genes, also referred to herein as “selection markers,” can include, for example, a hygromycin resistance gene.

[180] In some embodiments, a unique sequence is embedded into the genome of a genetically modified organism described herein using CRISPR methods for identification purposes. In some embodiments, this is referred to as a marker or marker sequence. In some embodiments this is referred to as a watermark sequence. In some embodiments, this is referred to as an intergenic sequence, or a portion thereof. In some embodiments, this is referred to as an intergenic watermark sequence. In some embodiments, this is referred to as barcoding. In some cases, the exogenous nucleic acid can include a barcode. The unique sequence is embedded into the genome of a genetically modified organism described herein using CRISPR methods for identification purposes. In some embodiments, this is referred to as a marker or marker sequence. In some embodiments this is referred to as a watermark sequence. In some embodiments, this is referred to as an intergenic sequence, or a portion thereof. In some embodiments, this is referred to as an intergenic watermark sequence. In some embodiments, this is referred to as barcoding. A barcode can comprise a non-natural sequence. In some embodiments, the barcode can be used to identify transgenic organisms via genotyping. In some embodiments, the exogenous nucleic acid can include a selectable marker, such as an antibiotic resistance gene. Selectable marker genes can include, for example, a hygromycin resistance gene. In some embodiments, this can be accomplished using resistance vector sequences like a resistance vector sequence shown in

TABLE 5A

TABLE 5A. Resistance vector and related gene sequences

Table 5B. Exemplary Terminator Sequences

[181] In some embodiments, a hygromycin resistance gene is used. In some embodiments, the hygromycin resistance gene sequence is SEQ ID NO. 302. In some embodiments, the sequence encoding the marker can be incorporated into the genetically modified cell or organism, for instance a fungal cell, yeast cell or plant cell as described herein. In some cases, a marker serves as a selection or screening device may function in a regenerable genetically modified organism to produce a compound that would confer upon a tissue in said organism resistance to an otherwise toxic compound. In some embodiments, the incorporated sequence encoding the marker may by subsequently removed from the transformed genome. Removal of a sequence encoding a marker may be facilitated by the presence of direct repeats before and after the region encoding the marker. In some embodiments, the marker sequence is followed by a protospacer adjacent motif (PAM), in order to provide appropriate cleavage by a Cas nuclease.

[182] In some embodiments, the exogenous nucleic acid can be introduced into the genetically modified organism by transformation or transfection.

[183] Transformation appropriate transformation techniques can include but are not limited to: electroporation of fungi protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of cells; microprojectile bombardment of cells; vacuum infiltration; and Agrobacterium tumeficiens mediated transformation. Transformation can mean introducing a nucleotide sequence into a cell in a manner to cause stable or transient expression of the sequence.

[184] Following transformation, fungi or other organisms can be selected using a dominant selectable marker incorporated into, for example, the transformation vector. In certain embodiments, such marker confers antibiotic or herbicide resistance on the transformed fungi or other organisms, and selection of transformants can be accomplished by exposing the fungi and other organisms to appropriate concentrations of the antibiotic or herbicide. In some embodiments, a ccdb negative selection marker is used. In some embodiments the ccdb negative selection marker is prepared by transforming a ccdb sensitive E. coli strain, e.g., DH5a. After transformed fungi or other organisms are selected and grown to maturity, those fungi and other organisms showing a modified trait are identified. The modified trait can be any of those traits described above. Additionally, expression levels or activity of the polypeptide or polynucleotide described herein can be determined by analyzing mRNA expression, using Northern blots, RT- PCR, RNA seq or microarrays, or protein expression using immunoblots or Western blots or gel shift assays.

[185] Suitable methods for transformation of fungal or other cells for use with the current disclosure can include virtually any method by which a nucleic acid can be introduced into a cell, such as by direct delivery of DNA such as by PEG-mediated transformation of protoplasts, by desiccation/inhibition-mediated DNA uptake, by electroporation, by agitation with silicon carbide fibers, by Agrobacterium-mediated transformation and by acceleration of DNA coated particles. Through the application of techniques such as these, the cells of virtually any fugus species may be stably transformed, and these cells developed into transgenic fungi.

[186] Methods of introducing an exogenous nucleic acid into a cell of an organism are generally known to the skilled artisan but may include the use of homology arms. In other instances, the exogenous nucleic acid can be randomly inserted into a genome of a target organism.

Agrobacterium-Mediated Transformation [187] Agrobacterium-mediated transfer can be used to introduce an exogenous nucleic acid into an organism selected for genetic modification, such as a fungal cell. In some instances, the exogenous nucleic acid can be introduced into whole fungal tissues, thereby by passing the need for regeneration of an intact fungus from a protoplast. The use of agrobacterium-mediated transformation can be used to integrate one or more vectors into the genetically modified organisms, including vectors or sequences encoding gene-editing systems, such as CRISPR systems or donor sequences.

[188] This disclosure includes advances in vectors for agrobacterium-mediated gene transfer by providing improved the arrangement of genes and restriction on sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. In some embodiments, a vector can have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for purposes described herein. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations.

[189] In some embodiments, a fungal cell, yeast cell, plant cell, may be modified using electroporation. To effect transformation by electroporation, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly. In some cases, electroporation may comprise 2 pulses, 3 pulses, 4 pulses, 5 pulses 6 pulses, 7 pulses, 8 pulses, 9 pulses, or 10 or more pulses. In some embodiments, protoplasts of fungi and/or plants may be used for electroporation transformation.

[190] Another method for delivering transforming DNA segments to fungal cells and cells derived from other organisms is microprojectile bombardment. In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, and preferably, gold. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. In some embodiments, DNA-coated particles may increase the level of DNA delivery via particle bombardment. For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells that can be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. In some cases, a starting cell density for genomic editing may be varied to optimize editing efficiency and/or cell viability. [191] In some embodiments, fungi, yeast or plants of the present disclosure can be used to produce new plant varieties. In some embodiments, the plants are used to develop new, unique and superior varieties or hybrids with desired phenotypes. In some embodiments, selection methods, e.g., molecular marker assisted selection, can be combined with breeding methods to accelerate the process. In some embodiments, a method comprises (i) crossing any organism provided herein comprising the expression cassette as a donor to a recipient organism line to create a FI population, (ii) selecting offspring that have expression cassette. Optionally, the offspring can be further selected by testing the expression of the gene of interest. In some embodiments, complete chromosomes of a donor organism are transferred. For example, the transgenic organism with an expression cassette can serve as a male or female parent in a cross pollination to produce offspring by receiving a transgene from a donor thereby generating offspring having an expression cassette. In a method for producing organisms having the expression cassette, protoplast fusion can also be used for the transfer of the transgene from a donor to a recipient. Protoplast fusion is an induced or spontaneous union, such as a somatic hybridization, between two or more protoplasts (cells in which the cell walls are removed by enzymatic treatment) to produce a single bi- or multi -nucleate cell. In some embodiments, mass selection can be utilized. In mass selection, desirable individual plants are chosen, harvested, and the seed composited without progeny testing to produce the following generation. Since selection is based on the maternal parent only, and there is no control over pollination, mass selection amounts to a form of random mating with selection. As stated herein, the purpose of mass selection is to increase the proportion of superior genotypes in the population.

[192] This disclosure provides gene editing systems for genetically modifying organisms. The gene editing system can be selected form the group consisting of a CRISPR system, TALEN, Zinc Finger, transposon-based, ZEN, meganuclease, Mega-TAL, and any combination thereof. In some embodiments, the gene editing system is directed to a target of interest by a guide polynucleotide. In some embodiments, the gene editing system involves an endonuclease or a nuclease or a polypeptide encoding a nuclease can be from a CRISPR (clustered regularly interspaced short palindromic repeats) system. An endonuclease or a nuclease or a polypeptide encoding a nuclease can be a Cas or a polypeptide encoding a Cas.

[193] CRISPR can refer to a family of DNA repeats found in certain bacterial genomes. In some instances, the CRISPR protein can include a Cas9 endonuclease, which can form a complex with a crRNA/tracrRNA hybrid to recognize, bind, and ultimately cleave foreign DNA at a target site. The crRNA/tracrRNA hybrid can be designed as a single guide RNA (gRNA). For example, any one or more of the guide RNAs, or targets thereof, are shown in TABLE 9 through TABLE 16. In some embodiments, the gRNA sequence is followed by a protospacer adjacent motif (PAM), in order to provide appropriate cleavage by a Cas nuclease.

[194] The recognition of a target DNA target region can depend on a protospacer adjacent motif (PAM) which can be located at the 3 '-terminus of a 20 bp target sequence. Once the CRISPR complex (e.g., Cas9 and associated guide RNA) recognizes the target DNA sequence, the CRISPR complex can generate a double strand break (DSB) at the DNA target locus. In some instances, one of two cellular DNA repair mechanisms, non-homologous end joining (NHEJ) and homologous recombination (HR), can play a role in precise genome editing and gene manipulation. For example, NHEJ, which is sometimes regarded as an error-prone repair mechanism that generates either short insertions or deletions of nucleotides in close proximity to the DSB site(s), can be used. If these short insertions or deletions exist in a gene coding region, or within a portion of the promoter involved in recruiting proteins involved in transcription, the function of the endogenous gene, for example a gene encoding psilocybin phosphatase, can be disrupted. Consequently, this procedure can be used for generating gene mutations. In other embodiments, a homology independent targeted integration (HITI) strategy can be used which allows fragments (e.g., exogenous nucleic acids) to be integrated into the genome by NHEJ repair.

[195] Various versions of CRISPR systems can be used. In some instances, the CRISPR system can be introduced into the genome of a target organism using Agrobacterium tumefaciens-mediated transformation. When the expression of Cas protein and guide RNA can be under the control of either a constitutive or inducible promoter. For example, in some embodiments, the Cas protein is under the control of a GDP gene protomer, while the guide RNA is under the control of a U6 gene promoter. In some embodiments, the guide RNA is inserted directly downstream of a P. cubensis U6 promoter and directly upstream of the guide RNA scaffold sequence. In some instances, the Cas protein is optimized for use in a fungal cell.

[196] The CRISPR system proteins disclosed herein may comprise one or more modifications. The modification may comprise a post-translational modification. The modification of the target nucleic acid may occur at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids away from the either the carboxy terminus or amino terminus end of the CRISPR system protein. The modification of the CRISPR system protein may occur at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids away from the carboxy terminus or amino terminus end of the CRISPR system protein. The modification may occur due to the modification of a nucleic acid encoding a CRISPR system protein. In some embodiments, SUMO refers to small ubiquitin-like modifier, Exemplary modifications can comprise methylation, demethylation, acetylation, deacetylation, ubiquitination, deubiquitination, deamination, alkylation, depurination, oxidation, pyrimidine dimer formation, transposition, recombination, chain elongation, ligation, glycosylation. Phosphorylation, dephosphorylation, adenylation, deadenylation, SUMOylation, deSUMOylation, ribosylation, deribosylation, myristoylation, remodelling, cleavage, oxidoreduction, hydrolation, and isomerization. The CRISPR system can comprise a modified form of a wild type exemplary CRISPR. The modified form of the wild type exemplary CRISPR system can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the CRISPR system.

[197] Genetic modifications of the disclosure can include substitutions, additions, and deletions, or any combination thereof. In some instances, the CRISPR system can target a nucleic acid. The CRISPR system can target DNA. In some instances, the CRISPR system comprises nickase activity. In some embodiments, the CRISPR system is modified to target a nucleic acid but is enzymatically inactive (e.g., does not have endonuclease or nickase activity). In some embodiments, simply by targeting an enzymatically inactive CRISPR system to a target nucleic acid the expression of one or more alkaloids is impacted. For example, targeting an enzymatically inactive CRISPR system to a target nucleic acid may function to prevent or displace a transcription factor that would otherwise be present thereby influencing the expression of a gene product involved in alkaloid production.

[198] In some embodiments, the CRISPR system can be active at temperatures suitable for growth and culture of a fungus or fungal cells, such as for example and without limitation, about 20 degrees Celsius to about 35 degrees Celsius, preferably about 23 degrees Celsius to about 32 degrees Celsius, and most preferably about 25 degrees Celsius to about 28 degrees Celsius.

[199] Accordingly, methods and compositions of the disclosure can be used at temperatures suitable for growth and culture of a fungus or fungal cells, such as for example and without limitation, 20 degrees Celsius to about 35 degrees Celsius, preferably about 23 degrees Celsius to about 32 degrees Celsius, and most preferably about 25 degrees Celsius to about 28 degrees Celsius.

[200] In some embodiments, the gene editing system is provided on a vector. For example, a non-replicating vector, such as, a viral vector. In other embodiments, the gene editing system is provided in a complex wherein the nucleic acid-targeting nucleic acid is pre-associated with a CRISPR/Cas protein. In some embodiments, the gene editing system is provided as part of an expression cassette on a suitable vector, configured for expression of a CRISPR system in a desired host cell (e.g., a fungal cell or a fungal protoplast). The vector may allow transient expression of a CRISPR/Cas protein. Alternatively, the vector may allow the expression cassette and/or CRISPR system to be stably maintained in the host cell, such as for example and not limitation, by integration into the host cell genome, including stable integration into the genome. In some embodiments, the host cell is an ancestral cell, thereby providing heritable expression of a CRISPR/Cas protein.

[201] This disclosure provides systems, compositions, and methods for genetically modifying a cell of an organism so as to produce one or more desirable alkaloids. An exemplary cell includes a fungal cell, such as a fungal protoplast. In some embodiments, the genetic modification is produced using a gene editing system.

[202] A gene editing (also called genome editing) system refers to a group of technologies that give the ability to change an organism's DNA. Many genome editing systems are based on bacterial nucleases. The systems, compositions, and methods described herein take advantage of genome editing systems to make targeted edits in an organism’s genome and thereby produce one or more alkaloids that are of interest. To that end, the genome editing systems as used herein can possess programmable nucleases. In some embodiments, the genome editing system comprises a zinc- finger nuclease (ZFN). A zinc finger nuclease is an artificial endonuclease that can comprise a designed zinc finger protein (ZFP) fused to a cleavage domain, such as, a FokI restriction enzyme. In some embodiments, the genome editing system comprises a transcription activator-like effector nuclease (TALEN). TALENs are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. In some embodiments, the genome editing system is a meganuclease. In some embodiments, a gene editing system is used in incorporate an exogenous nucleic acid into a fungal, wherein incorporation of the exogenous nucleic acid results in a genetic modification that modulates production of an alkaloid. In some embodiments, the exogenous nucleic acid comprises a sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or is 100% identical to one of the sequences listed in Table 2, Table 3A, or Table 3B. In some embodiments, the gene editing system is a CRISPR system, such as the CRISPR-Cas9 endonuclease system.

[203] CRISPR (clustered regularly interspaced short palindromic repeats) can refer to a family of DNA repeats found in certain bacterial genomes. In some instances, the CRISPR protein can include a Cas9 endonuclease, which can form a complex with a crRNA/tracrRNA hybrid to recognize, bind, and ultimately cleave foreign DNA at a target site. The crRNA/tracrRNA hybrid can be designed as a single guide RNA (gRNA). The guide RNAs can comprise a sequence that is capable of binding to any one of the target sequences shown in Tables 9-17. In some embodiments, the target sequence is followed by a protospacer adjacent motif (PAM), in order to provide appropriate cleavage by a Cas nuclease.

[204] In some embodiments, the guide RNA binds to a gene comprising a target sequence shown in Tables 9-17, and SEQ ID NOS: 60-116. In some embodiments, the guide RNA within about 100 bases, about 75 bases, about 50 bases, about 25 bases, about 5 bases, or 1 base of the sequence comprising one of SEQ ID NOS: 60-116. In some embodiments, the guide RNA binds to the gene at a loci at least partially overlapping the sequence comprising one of SEQ ID NOS: 60-116. In some embodiments, the at least one guide polynucleotide comprises a targeting sequence that is complementary to one of SEQ ID NOS: 60-116. In some embodiments, the at least one guide polynucleotide comprises a targeting sequence that binds to one of SEQ ID NOS: 60-116.

[205] The recognition of a target DNA target region can depend on a protospacer adjacent motif (PAM) which can be located at the 3'-terminus of a 20 bp target sequence, e.g., see Tables 9-17. Once the CRISPR complex (e.g., Cas9 and associated guide RNA) recognizes the target DNA sequence, the CRISPR complex can generate a double strand break (DSB) at the DNA target locus. In some instances, one of two cellular DNA repair mechanisms, non-homologous end joining (NHEJ) and homologous recombination (HR), can play a role in precise genome editing and gene manipulation. For example, NHEJ, which is sometimes regarded as an error-prone repair mechanism that generates either short insertions or deletions of nucleotides in close proximity to the DSB site(s), can be used. If these short insertions or deletions exist in a gene coding region, or within a portion of the promoter involved in recruiting proteins involved in transcription, the function of the endogenous gene, for example a gene encoding psilocybin phosphatase, can be disrupted. Consequently, this procedure can be used for generating gene mutations. In other embodiments, a homology independent targeted integration (HITI) strategy can be used which allows fragments (e.g., exogenous nucleic acids) to be integrated into the genome by NHEJ repair.

[206] Various versions of CRISPR systems can be used. In some instances, the CRISPR system can be introduced into the genome of a target organism using Agrobacterium tumefaciens- mediated transformation. When the expression of Cas protein and guide RNA can be under the control of either a constitutive or inducible promoter. For example, in some embodiments, the Cas protein is under the control of a GDP gene protomer, while the guide RNA is under the control of a U6 gene promoter. In some embodiments, the guide RNA is inserted directly downstream of a P. cubensis U6 promoter and directly upstream of the guide RNA scaffold sequence. In some instances, the Cas protein is optimized for use in a fungal cell. [207] In some embodiments, an endonuclease system that is used to genetically modified an organism described herein comprises a CRISPR enzyme and a guide nucleic acid that hybridizes with a target sequence in, or adjacent to the gene or the promoter or enhancer associated therewith. In some cases, a target sequence can be at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides in length.

[208] A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and/or within a complement of a target sequence. In some embodiments, a target sequence is at least about 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides in length. In some embodiments, a target sequence is at most 17 nucleotides in length. In some aspects, a target can be selected from a sequence comprising homology from about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% to any one of SEQ ID NOS: 1 to 18, or 89-99. In some embodiments, the target is a psilocybin synthase gene.

[209] The CRISPR enzyme can be guided by a guide polynucleotide, which can be DNA or RNA. A guide polynucleotide acid can be single stranded or double stranded. In some cases, a guide polynucleotide contains regions of single stranded areas and double stranded areas, guide polynucleotide can also form secondary structures. As used herein, the term “guide RNA (gRNA),” and its grammatical equivalents can refer to an RNA which can be specific for a target DNA and can form a complex with a Cas protein. A guide RNA can comprise a guide sequence, or spacer sequence, that specifies a target site and guides an RNA/Cas complex to a specified target DNA for cleavage. For example, a guide RNA can target a CRISPR complex to a target gene or portion thereof and perform a targeted double strand break. The target gene can be a gene listed in Tables 1 and 2. Site-specific cleavage of a target DNA occurs at locations determined by both 1) base-pairing complementarity between a guide RNA and a target DNA (also called a protospacer) and 2) a short motif in a target DNA referred to as a protospacer adjacent motif (PAM). In some cases, gRNAs can be designed using an algorithm which can identify gRNAs located in early exons within commonly expressed transcripts.

[210] In some embodiments, a guide polynucleotide hybridizes with a target sequence, which can be 7 nucleotides in length. In some embodiments, a target sequence comprises a portion of at least one of SEQ ID NOS: 1-18 or 89-99, a gene or a regulator element of a gene selected from Table 1 or Table 2. In some embodiments, a guide nucleic acid can be chemically modified. In an embodiment, a guide polynucleotide is a single guide RNA (sgRNA). In an embodiment, a guide nucleic acid can be a chimeric single guide comprising RNA and DNA. In some cases, a CRISPR enzyme can comprise or be a Cas protein or variant or derivative thereof. In some cases, a Cas protein comprises Cast, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, CasSt, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, CseSe, Csci, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csrn4, Csm5, Csm6, Cmr, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csf 1, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, C2cl, C2c2, C2c3, Cpfl, CARF, DinG, homologues thereof, or modified versions thereof. In some cases, a Cas protein can be a Cas9. In some cases, Cas9 is a modified Cas9 that binds to a canonical PAM. In some cases, Cas9 recognizes a non-canonical PAM. In some cases, a guide polynucleotide binds a target sequence 3-10 nucleotides from a PAM. In some cases, a CRISPR enzyme coupled with a guide polynucleotide can be delivered into a genetically modified organism as an RNP. In some cases, a CRISPR enzyme coupled with a guide polynucleotide can be delivered into a genetically modified organism by a mRNA encoding the CRISPR enzyme and the guide polynucleotide.

[2H] The CRISPR system may be used to introduce a double-stranded break into a target nucleic acid, e.g., genomic DNA, of an organism, such as a fungal protoplast. The double-stranded break can stimulate a fungal cell’s endogenous DNA-repair pathway (e.g., HR, NHEJ, A-NHEJ, or MMEJ). NHEJ can repair cleaved target nucleic acid without the need for a homologous template. This can result in desired deletions of the target nucleic acid. Homologous recombination (HR) can occur with a homologous template. The homologous template can comprise sequences that are homologous to sequences flanking the target nucleic acid cleavage site. After a target nucleic acid is cleaved by CRISPR/Cas9, the site of cleavage can be destroyed (e.g., the site may not be accessible for another round of cleavage with the original nucleic acid-targeting nucleic acid and CRISPR/Cas9).

[212] A CRISPR system can comprise a nucleic acid-binding domain, e.g., a guide polynucleotide. The nucleic acid-binding domain can comprise a region that contacts a nucleic acid. A nucleic acid-binding domain can comprise a nucleic acid. A nucleic acid-binding domain can comprise DNA. A nucleic acid-binding domain can comprise single stranded DNA. Examples of nucleic acid-binding domains can include, but are not limited to, a helix-tum-helix domain, a zinc finger domain, a leucine zipper (bZIP) domain, a winged helix domain, a winged helix turn helix domain, a helix-loop-helix domain, an HMG-box domain, a Wor3 domain, an immunoglobulin domain, a B3 domain, and a TALE domain. A nucleic acid-binding domain can be a domain of a CRISPR system protein. A CRISPR system protein can be a eukaryotic CRISPR system or a prokaryotic CRISPR. A CRISPR system protein can bind RNA or DNA, or both RNA and DNA. In some embodiments, a CRISPR system protein binds a DNA and cleaves the DNA. In some instances, the CRISPR system protein binds a double-stranded DNA and cleaves a double- stranded DNA. In some instances, two or more nucleic acid-binding domains can be linked together. Linking a plurality of nucleic acid-binding domains together can provide increased polynucleotide targeting specificity. Two or more nucleic acid-binding domains can be linked via one or more linkers. The linker can be a flexible linker. Linkers can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or more nucleotides or amino acids in length. The linker domain may comprise glycine and/or serine, and in some embodiments may consist of or may consist essentially of glycine and/or serine. Linkers can be a nucleic acid linker which can comprise nucleotides. A nucleic acid linker can link two DNA- binding domains together. A nucleic acid linker can be at most 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. A nucleic acid linker can be at least 5, 10, 15, 30, 35, 40, 45, or 50 or more nucleotides in length. Nucleic acid-binding domains can bind to nucleic acid sequences. Nucleic acid binding domains can bind to nucleic acids through hybridization. Nucleic acidbinding domains can be engineered (e.g., engineered to hybridize to a sequence in a genome). A nucleic acid-binding domain can be engineered by molecular cloning techniques (e.g., directed evolution, site-specific mutation, and rational mutagenesis). A CRISPR system can comprise a nucleic acid-cleaving domain. The nucleic acid-cleaving domain can be a nucleic acid-cleaving domain from any nucleic acid-cleaving protein. The nucleic acid-cleaving domain can originate from a nuclease. Suitable nucleic acid-cleaving domains include the nucleic acid-cleaving domain of endonucleases (e.g., AP endonuclease, RecBCD enonuclease, T7 endonuclease, T4 endonuclease IV, Bal 31 endonuclease, Endonuclease I (endo I), Micrococcal nuclease, Endonuclease II (endo VI, exo III)), exonucleases, restriction nucleases, endoribonucleases, exoribonucleases, RNases (e.g., RNAse I, II, or III). A nucleic acid-binding domain can be a domain of a CRISPR system protein. A CRISPR system protein can be a eukaryotic CRISPR system or a prokaryotic CRISPR/CasX. A CRISPR system protein can bind RNA or DNA, or both RNA and DNA. A CRISPR system protein can cleave RNA, or DNA, or both RNA and DNA. In some embodiments, a CRISPR system protein binds a DNA and cleaves the DNA. In some embodiments, the CRISPR system protein binds a double-stranded DNA and cleaves a doublestranded DNA. In some embodiments, the nucleic acid-cleaving domain can originate from the Fokl endonuclease.

[213] A CRISPR system can comprise a plurality of nucleic acid-cleaving domains. Nucleic acidcleaving domains can be linked together. Two or more nucleic acid-cleaving domains can be linked via a linker. In some embodiments, the linker can be a flexible linker as described herein. Linkers can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or more amino acids in length. In some embodiments, a CRISPR system can comprise the plurality of nucleic acid-cleaving domains. CRISPR system can introduce double-stranded breaks in nucleic acid, (e.g., genomic DNA). The double-stranded break can stimulate a cell's endogenous DNA-repair pathways (e.g., homologous recombination and non-homologous end joining (NHEJ) or alternative nonhomologues end joining (A-NHEJ)). NHEJ can repair cleaved target nucleic acid without the need for a homologous template. This can result in deletions of the target nucleic acid. Homologous recombination (HR) can occur with a homologous template. The homologous template can comprise sequences that are homologous to sequences flanking the target nucleic acid cleavage site. After a target nucleic acid is cleaved by a CRISPR system the site of cleavage can be destroyed (e.g., the site may not be accessible for another round of cleavage with the original nucleic acid targeting nucleic acid and CRISPR/Cas9).

[214] In some embodiments, this disclosure provides a genetic modification that can involve introducing an exogenous nucleic acid into an organism and/or performing a gene deletion/disruption in the organism. In some instances, this can be accomplished using homologous recombination (HR), wherein selective markers conferring resistance to, for example, an antifungal compound, e.g., hygromycin or neomycin, can be used to replace or integrate within the target locus. While some fungi, e.g., Saccharomyces cerevisiae, may have a relatively high HR efficiency, gene disruption can be difficult for many other fungal organisms due to a low HR efficiency. Provided here are efficient, rapid, powerful, and economical gene manipulation tools such as CRISPR technology, which as described in certain embodiments herein, is optimized for use on fungal organisms. This technology can be used to enhance the efficiency of gene manipulation and integration of, for example, one or more exogenous nucleic acids into a fungal cell.

[215] In some embodiments, homologous recombination can insert an exogenous polynucleotide sequence into the target nucleic acid cleavage site. The exogenous polynucleotide can comprise any sequence of TABLE 2, or TABLE 3A-3B. An exogenous polynucleotide sequence can be called a donor polynucleotide or a donor sequence. In some embodiments of compositions and methods of the disclosure, the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide can be inserted into the target nucleic acid cleavage site. A donor polynucleotide can be an exogenous polynucleotide sequence. A donor polynucleotide can be a sequence that does not naturally occur at the target nucleic acid cleavage site. A vector can comprise a donor polynucleotide. The modifications of the target DNA due to NHEJ and/or HR can lead to, for example, mutations, deletions, alterations, integrations, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, and/or gene mutation. The process of integrating non-native nucleic acid into genomic DNA can be referred to as genome engineering.

[216] The CRISPR system proteins disclosed herein may comprise one or more modifications. The modification may comprise a post-translational modification. The modification of the target nucleic acid may occur at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids away from the either the carboxy terminus or amino terminus end of the CRISPR system protein. The modification of the CRISPR system protein may occur at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acids away from the carboxy terminus or amino terminus end of the CRISPR system protein. The modification may occur due to the modification of a nucleic acid encoding a CRISPR system protein. Exemplary modifications can comprise methylation, demethylation, acetylation, deacetylation, ubiquitination, deubiquitination, deamination, alkylation, depurination, oxidation, pyrimidine dimer formation, transposition, recombination, chain elongation, ligation, glycosylation. Phosphorylation, dephosphorylation, adenylation, deadenylation, SUMOylation, deSUMOylation, ribosylation, deribosylation, myristoylation, remodelling, cleavage, oxidoreduction, hydrolation, and isomerization. The CRISPR system can comprise a modified form of a wild type exemplary CRISPR. The modified form of the wild type exemplary CRISPR system can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the CRISPR system.

[217] Genetic modifications of the disclosure can include substitutions, additions, and deletions, or any combination thereof. In some instances, the CRISPR system can target a nucleic acid. The CRISPR system can target DNA. In some instances, the CRISPR system comprises nickase activity. In some embodiments, the CRISPR system is modified to target a nucleic acid but is enzymatically inactive (e.g., does not have endonuclease or nickase activity). In some embodiments, simply by targeting an enzymatically inactive CRISPR system to a target nucleic acid the expression of one or more alkaloids is impacted. For example, targeting an enzymatically inactive CRISPR system to a target nucleic acid may function to prevent or displace a transcription factor that would otherwise be present thereby influencing the expression of a gene product involved in alkaloid production.

[218] In some embodiments, the CRISPR system can be active at temperatures suitable for growth and culture of a fungus or fungal cells, such as for example and without limitation, about 20 degrees Celsius to about 35 degrees Celsius, preferably about 23 degrees Celsius to about 32 degrees Celsius, and most preferably about 25 degrees Celsius to about 28 degrees Celsius. [219] Accordingly, methods and compositions of the disclosure can be used at temperatures suitable for growth and culture of a fungus or fungal cells, such as for example and without limitation, 20 degrees Celsius to about 35 degrees Celsius, preferably about 23 degrees Celsius to about 32 degrees Celsius, and most preferably about 25 degrees Celsius to about 28 degrees Celsius.

[220] In some embodiments, the gene editing system is provided on a vector. For example, a non-replicating vector, such as, a viral vector. In other embodiments, the gene editing system is provided in a complex wherein the nucleic acid-targeting nucleic acid is pre-associated with a CRISPR/Cas protein. In some embodiments, the gene editing system is provided as part of an expression cassette on a suitable vector, configured for expression of a CRISPR system in a desired host cell (e.g., a fungal cell or a fungal protoplast).

Genetic Engineering using Homologous Directed Repair and Methods for Introducing Fungal DNA

[221] In some embodiments, an exogenous nucleic acid can be integrated into the genome of a genetically modified organism described herein by homologous recombination. Homologous recombination permits site specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the genome of the genetically modified organism. One method that can lead to precise sequence alterations at specified genomic locations is by using a homologous directed repair (HDR) method (FIG. 19). Designed HDR donor templates can contain sequences homologous to the specific sequence flanking the cut site, referred to herein as “homology arms”. In some embodiments, -nts refers to nucleotides. In some embodiments, a homology arm is at least: 10-nts, 15-nts, 20-nts, 25-nts, 30- nts, 35-nts, 40-nts, 50-nts, 55-nts, 60-nts, 65-nts, or 70-nts, 80-nts, 90-nts, 100-nts, 110-nts, 120- nts, 130-nts, or 140-nts.

HDR for self-replicating plasmids and protoplasts

[222] In some embodiments HDR is carried out using self-replicating plasmids and protoplasts. In some embodiments, a PsiD gene locus is targeted. In some embodiments, targeting a PsiD locus produces an edited, non-genetically modified psilocybe cubensis fungus that is genetically engineered. In some embodiments, targeting a PsiD locus in a genetic engineering process results in overexpressing PsiD. In some embodiments, the targeted PsiD gene locus undergoes HDR to produce a gene-edited Psilocybe cubensis fungus that overexpress, underexpresses, or does not express PsiD. In some embodiments, this HDR method results in a non-genetically modified fungus comprising a genetic modification. In some embodiments, B-AMA1 replication origin - containing plasmid is allowed to replicate in a fungal cell without being integrated into the genome of the fungal cell. In some embodiments, the B-AMA1 replication origin-containing plasmid further comprises a hygromycin resistance gene sequence. In some embodiments, the B-AMA1 replication origin-containing plasmid further comprises a Cas endonuclease. In some embodiments, the Cas endonuclease is an SpCas enzyme. In some embodiments, the Cas endonuclease is an optimized SpCas enzyme sequence. In some embodiments, the optimized SpCas enzyme comprises a sequence with a percent identity of about: 80%, 85%, 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% to any one of SEQ ID NOs: 640-647. In some embodiments, the optimized SpCas enzyme comprises a sequence is any one of SEQ ID NOs: 640-647. In some embodiments, a hygromycin-resistant cassette and transfected protoplasts can then be selected in the presence of hygromycin until only until HDR is completed. Antibiotic selection is then removed and the plasmid is no longer part of the protoplast. This produces a gene- edited fungus. In some embodiments, the resistance cassette can be re-used to target a subsequent locus in the genome as needed. In some embodiments, Golden Gate cloning strategy is used to produce the gene-edited fungus. In some embodiments, the plasmid comprises an SpCas9 enzyme. In some embodiments, the plasmid comprises a codon optimized SpCas9 enzyme. Exemplary plasmids comprising a SpCas9 enzyme are shown in TABLE 6A. In some embodiments, the plasmid comprises an SpCas enzyme comprising any one of SEQ ID NOs: 640-647. In some embodiments, the plasmid comprises an SpCas enzyme comprising any one of SEQ ID NOs: 640- 647. In some embodiments, the SpCas enzyme is selected from the group consisting of any one of SEQ ID NOs: 640-647. In some embodiments, the Cas enzyme used is a SpCas9 sequence Utsilago maydis codon-optimized on the backbone.

TABLE 6A. Exemplary Sequences comprising SpCas9 for HDR methods

Table 6B. Exemplary Cas sequences

[223] In some embodiments, the HDR cassette and the guide RNAs of a gene described herein are cloned into the plasmid. In some embodiments, the HDR cassette and the guide RNAs of a PsiD gene described herein are cloned into the plasmid.

[224] In some embodiments, pCambrial300 plasmid with an introduced B-AMA1 sequence is used in an HDR method described herein. In some embodiments a pCambrial300 comprising an introduced B-AMA1 sequence plasmid can become self-replicating. In some embodiments different spCas9 variants are used in an HDR method described herein. In some embodiments, a plasmid described herein comprises an spCas9 variant. In some embodiments, an HDR cassette and a PsiD gene guide RNA can be cloned into the plasmid. In some embodiments, B-AMA1 replication origin and a Cas9 variant can be cloned into the plasmid. In some embodiments, an HDR cassette and a PsiD gene guide RNA, the B-AMA1 replication origin and a Cas9 variant can be cloned into the plasmid. In some embodiments, an entry vector is assembled with a final plasmid backbone for protoplast transformation is in a Magic Gate reaction using a Bsal restriction enzyme. In some embodiments, the entry vector is HDRPsiDguideMGRiboF with a sequence comprising: CACCtgggagCTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCCTCCCAACACTT GATCATGC. In some embodiments, the entry vector is HDRPsiDguideMGRiboR with a sequence comprising:

AAACGCATGATCAAGTGTTGGGAGGACGAGCTTACTCGTTTCGTCCTCACGGACTCA TCAGctccca. In some embodiments, a PsiD guide RNA is introduced into a minor groove binding (MGB) ribozyme backbone. In some embodiments, this components plasmid comprising the sequence: GACGCTGTGGATCAAGCAACGCCACTCGCTCGCTCCATCGCAGGCTGGTCGCAGAC AAATTAAAAGGCGGCAAACTCGTACAGCCGCGGGGTTGTCCGCTGCAAAGTACAGA GTGATAAAAGCCGCCATGCGACCATCAACGCGTTGATGCCCAGCTTTTTCGATCCGA GAATCCACCGTAGAGGCGATAGCAAGTAAAGAAAAGCTAAACAAAAAAAAATTTCT GCCCCTAAGCCATGAAAACGAGATGGGGTGGAGCAGAACCAAGGAAAGAGTCGCG CTGGGCTGCCGTTCCGGAAGGTGTTGTAAAGGCTCGACGCCCAAGGTGGGAGTCTA GGAGAAGAATTTGCATCGGGAGTGGGGCGGGTTACCCCTCCATATCCAATGACAGA TATCTACCAGCCAAGGGTTTGAGCCCGCCCGCTTAGTCGTCGTCCTCGCTTGCCCCTC CATAAAAGGATTTCCCCTCCCCCTCCCACAAAATTTTCTTTCCCTTCCTCTCCTTGTC CGCTTCAGTACGTATATCTTCCCTTCCCTCGCTTCTCTCCTCCATCCTTCTTTCATCCA TCTCCTGCTAACTTCTCTGCTCAGCACCTCTACGCATTACTAGCCGTAGTATCTGAGC ACTTCTCCCTTTTATATTCCACAAAACATAACACAACCTTCACCtgggagCTGATGAGTC CGTGAGGACGAAACGAGTAAGCTCGTCCTCCCAACACTTGATCATGCGTTTTAGAGC TAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACC GAGTCGGTGCTTTTGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAA CATGCTTCGGCATGGCGAATGGGACTGAGAAACAGGTCGGAAGCCAATGGCCAGGA GCTCCTTGTAAAAAAATACTCCTTGGTCTATTAAGTTGCCCATTCTTTAGCAGGAGT GTGCAGACTATGTCCGTATCCACATGCCGCAACTGCAGATTCATAGGAGCTGTTGGG GATATTGGCATAGGATCCCATTGTTACGTACTATTTAATGACAAATACACGATCAAT TTCACCACTATTGTTCACTTCTACTGGTAGCTTAGACGTACTATTTCTCGTGGAATAG CCAGTACTTGCTCTTATATTGGCCGTCGCGAATTTCGGCGTCGACAACGAGCTACCA CATTTGTTCATGCCAGGCAGCTGAGGACTTGAAAGCCTTGAAATGCCGAAGGTAGT ATATCCCGCGTTCCTTTATCAGATTAGAACAAATGCCGTTCTATCATCTGGGTATACT TAGTCCTTTTGACCGGGGAAATATGTCACGTGCAAGGCGCTTTGGAAGCTTCCGACC (SEQ ID NO: 640).

[225] In some embodiments, a repair cassette described herein is cloned. In some embodiments, primers are used to amplify a homologous recombination (HR) of a PsiD gene. In some embodiments the primer is a primer listed in TABLE 7A-7C.

Table 7A. Exemplary Primers for left-flanking HR PsiD amplification - PsiD repair cassette cloning

Table 7B. Exemplary Primers for Right-flanking HR PsiD amplification - PsiD repair cassette cloning

[226] In some embodiments, a GPD-intron described herein will be amplified using a primer in TABLE 7C with a GPD:intron plasmid described herein.

Table 7C. Exemplary Primers for GPD-intron amplification - PsiD repair cassette cloning

[227] In some embodiments, a combination of primers from TABLES 7A-7C are used. In some embodiments the plasmid comprises a tRNA-gRNA-scaffold sequence. In some embodiments, the gRNA comprises a PsiD gene gRNA. In some embodiments, the tRNA sequence comprises: ACTAGATTCCCTTACGCCTTCCATCACCTGTCCGCACCCGGCCCCATCCCGCTTTCAA CCCCCCGCTCCGAGCCGGCACCGGAGCACACCCACCCAAACCGGTTCGATGGCGTA GTTGGTTATCGCATCTGTCTAACACACAGAAGGTCCTCAGTTCGAGCCTGGGTCGAA TCA. In some embodiments the gRNA sequence comprises: CTCCCAACACTTGATCATGC. In some embodiments the scaffold sequence comprises: gtttTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA GTGGCACCGAGTCGGTGCTTTTTTatgccacaacactggtggtacc. In some embodiments the tRNA sequence is:

ACTAGATTCCCTTACGCCTTCCATCACCTGTCCGCACCCGGCCCCATCCCGCTTTCAA CCCCCCGCTCCGAGCCGGCACCGGAGCACACCCACCCAAACCGGTTCGATGGCGTA GTTGGTTATCGCATCTGTCTAACACACAGAAGGTCCTCAGTTCGAGCCTGGGTCGAA TCA. In some embodiments the gRNA sequence is: CTCCCAACACTTGATCATGC. In some embodiments the scaffold sequence is: gtttTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAA GTGGCACCGAGTCGGTGCTTTTTTatgccacaacactggtggtacc. In some embodiments, tRNA- gRNA-scaffold sequence comprises:

ACTAGATTCCCTTACGCCTTCCATCACCTGTCCGCACCCGGCCCCATCCCGCTTTCAA CCCCCCGCTCCGAGCCGGCACCGGAGCACACCCACCCAAACCGGTTCGATGGCGTA GTTGGTTATCGCATCTGTCTAACACACAGAAGGTCCTCAGTTCGAGCCTGGGTCGAA TCACTCCCAACACTTGATCATGCgtttTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTatgccacaacactggtgg tacc. In some embodiments, tRNA-gRNA-scaffold sequence is: ACTAGATTCCCTTACGCCTTCCATCACCTGTCCGCACCCGGCCCCATCCCGCTTTCAA CCCCCCGCTCCGAGCCGGCACCGGAGCACACCCACCCAAACCGGTTCGATGGCGTA GTTGGTTATCGCATCTGTCTAACACACAGAAGGTCCTCAGTTCGAGCCTGGGTCGAA TCACTCCCAACACTTGATCATGCgtttTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTatgccacaacactggtgg tacc. In some embodiments, tRNA-gRNA-scaffold is merged together with an HDR repair cassette sequence and a B-AMA1 sequence. In some embodiments the HDR repair cassette sequence comprises:

ACTGGATTAGGTTGAAGAACCGGCGATCTGGGCAGACGCGCCACGCTCTGAGTACC TAAGGGTGTACTTAAACTGGATTAGGTTGAAGAACCGGCGATCTGGGCAGACGCGC CACGCTCTGAGTACCTAAGGGTGTACTTAAATTTATCACAGCTTGACGTTTGACCTG GAAGCTTGATTTACGCAAGGTTGGAACTTGCACCCCCCGGTCGAGCATCTCTCTCTA GTCATAGTTTATCTTTGTATAAATGGGGGCCTCAACGCAAGGCCGCAAAACTACTCC CAACTTTTATAACTCATTTCTGCTCCCAACACTTGATCAGAGGTCCGCAAGTAGATT GAAAGTTCAGTACGTTTTTAACAATAGAGCATTTTCGAGGCTTGCGTCATTCTGTGT CAGGCTAGCAGTTTATAAGCGTTGAGGATCTAGAGCTGCTGTTCCCGCGTCTCGAAT GTTCTCGGTGTTTAGGGGTTAGCAATCTGATATGATAATAATTTGTGATGACATCGA TAGTACAAAAACCCCAATTCCGGTCACATCCACCATCTCCGTTTTCTCCCATCTACAC ACAACAAGCTCATCGCCGTTTGTCTCTCGCTTGCATACCACCCAGCAGCTCACTGAT GTCGACTTGTAGaTGCAGGTGATACCCGCGTGCAACTCGGCGTACGTCGTTTTTATTC GCTGACTTCACCCGCTAATTACTATAACTTGAAAACACAGAGCAATAAGATCACTAT

GTCCTACTCCCGAGTCTTTGAGAAACATGGGATGGCTCTCTGTCAGCGATGCGGTCT ACAGCGAGTTCATAGGAGAGTTGGCTACCCGCGCTTCCAATCGAAATTACTCCAACG AGTTCGGCCTCATGCAACCTATCCAGGAATTCA. In some embodiments the HDR repair cassette sequence is:

ACTGGATTAGGTTGAAGAACCGGCGATCTGGGCAGACGCGCCACGCTCTGAGTACC TAAGGGTGTACTTAAACTGGATTAGGTTGAAGAACCGGCGATCTGGGCAGACGCGC CACGCTCTGAGTACCTAAGGGTGTACTTAAATTTATCACAGCTTGACGTTTGACCTG GAAGCTTGATTTACGCAAGGTTGGAACTTGCACCCCCCGGTCGAGCATCTCTCTCTA GTCATAGTTTATCTTTGTATAAATGGGGGCCTCAACGCAAGGCCGCAAAACTACTCC CAACTTTTATAACTCATTTCTGCTCCCAACACTTGATCAGAGGTCCGCAAGTAGATT GAAAGTTCAGTACGTTTTTAACAATAGAGCATTTTCGAGGCTTGCGTCATTCTGTGT CAGGCTAGCAGTTTATAAGCGTTGAGGATCTAGAGCTGCTGTTCCCGCGTCTCGAAT GTTCTCGGTGTTTAGGGGTTAGCAATCTGATATGATAATAATTTGTGATGACATCGA

TAGTACAAAAACCCCAATTCCGGTCACATCCACCATCTCCGTTTTCTCCCATCTACAC

sequence comprises: attaccgatcctcgatctttgtgcaagctagcccgcctcggcagcaacaaagcagccgagcaagaagcagtacttgccttctgaatcgtgaa tgggttacgttcttcaccgctgtgatcagcgaatcatgaatcaaatcatgagggcattgctgatcatgaatcaaatcatgagggcatttaaaaat tcagtctgagtcgtgagtagcaagtcggttctggatcggatggcattcatgaatcacagggtcgtgaatcatgaatgttcaagtccccttttctc gagaggctggtgggatcggtgcgaatcacgaatcatgattgtaattcattgagtgaaggagtttcgcagccacccacagtactagaatcacg aatgacaat (SEQ ID NO: 648). In some embodiments, the B-AMA1 sequence is: attaccgatcctcgatctttgtgcaagctagcccgcctcggcagcaacaaagcagccgagcaagaagcagtacttgccttctgaatcgtgaa tgggttacgttcttcaccgctgtgatcagcgaatcatgaatcaaatcatgagggcattgctgatcatgaatcaaatcatgagggcatttaaaaat tcagtctgagtcgtgagtagcaagtcggttctggatcggatggcattcatgaatcacagggtcgtgaatcatgaatgttcaagtccccttttctc gagaggctggtgggatcggtgcgaatcacgaatcatgattgtaattcattgagtgaaggagtttcgcagccacccacagtactagaatcacg aatgacaat (SEQ ID NO: 649). In some embodiments, a B-AMA1 sequence, and HDR repair cassette, and a tRNA-gRNA-scaffold are merged by an overlapping PCR method and inserted into a pMGA entry plasmid. In some embodiments the pMGA plasmid comprises SEQ ID: 309.

Microhomology mediated end joint methods

[228] In some embodiments, a method for double-stranded repair (DSB) is used to gene-edit a fungus. In some embodiments, the method of gene-editing used is microhomology mediated end joint (MMEJ), also referred to as alternative non-homologous end joint (A-NHEJ). MME J is similar to Homologous directed repair (HDR) involves end resection and rely on homologous sequence for DSB repair. The length of the homologues sequence used by MMEJ and HDR can be different. With MMEJ the homology flanking region ranges from 2-50bp while HDR uses between 500 to 5000bp. For MMEJ repair involves annealing the small sequences and gap filling by DNA polymerase theta near the DSB, resulting in small to large deletions and templated insertions. This approach involves the use of in vitro assembled Crispr Cas9 ribonucleoprotein (RNP) complexes and double stranded DNA template repair (35S promoter: Hygromycin:35S terminator) flanked with 35 bp microhomology sequence upstream and downstream regions of the DSB site that allows for precise homologous recombination after the Cas targeted cut. In some embodiments, the 35 bp microhomology sequence comprises: CTAATGAATATTAGCCAGTACGTCGCGTCGAACGA. In some embodiments, the 35 bp microhomology sequence comprises: TGCTAATTGGCAGTAGCACGATTTATCGTGTGCCG. In some embodiments, the 35 bp microhomology sequence is a guide RNA. In some embodiments, the 35 bp microhomology sequence functions as a guide RNA. In some embodiments, the 35 bp microhomology sequence is operably linked to a target locus sequence. In some embodiments, an MMEJ method for gene editing includes the use of a target locus sequence. In some embodiments, the target locus sequence comprises:

CTCGGCATATCGGCTATCATGCAATATTATTGGCTGGGCATCGACTCCGGTTTAAAA ACTCCATCGGACTTGTATCTTGCAATCCGGCTGTCACTGCCTTTTCCTTGCCCATCTT GAAGTTCGTCGGTTCCCGTTTTCTCCGAACAAGGATTTTGGGTAGTATGACGACAGA TGCATCATTACTTGTGCGAGCAAATCGGATTCCATTACTCATGGAGCGGGCGGCGCT AATGAATATTAGCCAGTACGTCGCGTCGAACGAAGGTCAACCATGTCCTATCGACA CTACAGTAATAGCTTCTTGCGCACACTAAGAAGTCTGGACACAAGAACCGTTGTATC ATTTGGATGGTTCCGCTCCCAGCCCGGTCAGCTGTCACAAGTGAGATCAAACCCGAC TTCGTCCGAGGGAAATGGCTTTCATATCAGTGAAAAGGTGTCAATATAAGTGAACAT TTCACCAATCTGCGGCACACGATAAATCGTGCTACTGCCAATTAGCAGTTGGCGTAG AGAAGCAATCGAGTAACTGATAGGAAAAGAAGGTATTATAAGGGAAAATTTAGAA CGTGGTTCCCTCACTAACCAACCTTTAGACAAGGCTCCTATCGTGCCGGGGTTCTTG TGCCCATTATAAGGTCGAAGGAGGAGACTATAGGCGGCAATGGAACCATCATCTTC ACACATCGAGGGTGTTCTGGAACAATTATGACGTTTCAATGAAGGGCATGCGATAC AAAAATGCAATGGTGACTTCAAGGTCAATATTGCCTTCATTTACAGAAACTGGTAAT CTATCTTCAATTGCAGCCAGAGAACTCCCCATCTGA. In some embodiments, the target locus sequence is:

CTCGGCATATCGGCTATCATGCAATATTATTGGCTGGGCATCGACTCCGGTTTAAAA ACTCCATCGGACTTGTATCTTGCAATCCGGCTGTCACTGCCTTTTCCTTGCCCATCTT GAAGTTCGTCGGTTCCCGTTTTCTCCGAACAAGGATTTTGGGTAGTATGACGACAGA TGCATCATTACTTGTGCGAGCAAATCGGATTCCATTACTCATGGAGCGGGCGGCGCT AATGAATATTAGCCAGTACGTCGCGTCGAACGAAGGTCAACCATGTCCTATCGACA CTACAGTAATAGCTTCTTGCGCACACTAAGAAGTCTGGACACAAGAACCGTTGTATC ATTTGGATGGTTCCGCTCCCAGCCCGGTCAGCTGTCACAAGTGAGATCAAACCCGAC TTCGTCCGAGGGAAATGGCTTTCATATCAGTGAAAAGGTGTCAATATAAGTGAACAT TTCACCAATCTGCGGCACACGATAAATCGTGCTACTGCCAATTAGCAGTTGGCGTAG AGAAGCAATCGAGTAACTGATAGGAAAAGAAGGTATTATAAGGGAAAATTTAGAA CGTGGTTCCCTCACTAACCAACCTTTAGACAAGGCTCCTATCGTGCCGGGGTTCTTG TGCCCATTATAAGGTCGAAGGAGGAGACTATAGGCGGCAATGGAACCATCATCTTC ACACATCGAGGGTGTTCTGGAACAATTATGACGTTTCAATGAAGGGCATGCGATAC AAAAATGCAATGGTGACTTCAAGGTCAATATTGCCTTCATTTACAGAAACTGGTAAT CTATCTTCAATTGCAGCCAGAGAACTCCCCATCTGA. In some embodiments, the target locus comprises a 35bp homology sequence comprising:

CTAATGAATATTAGCCAGTACGTCGCGTCGAACGA. In some embodiments, the target locus comprises a 35bp homology sequence comprising:

TGCTAATTGGCAGTAGCACGATTTATCGTGTGCCG. In some embodiments, an MMEJ method used herein comprises a zero blunt topo vector backbone, an enhanced 35S promoter, a hygromycin gene, and a 35S terminator. In some embodiments, an MMEJ method used herein has a repair template comprising:

AGTGTGCTGGAATTCGCCCTTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTC CTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAA GGTGGCACCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGC CTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAA AAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATAACATGGTG GAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCA AAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCA TTGCCCAGCTATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCT ACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGAC AGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGT TCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGA TGACGCACAATCCCACTATCCTTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTT CATTTGGAGAGGACACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCG AGCTTTCGCAGATCCCGGGGGGCAATGAGATATGAAAAAGCCTGAACTCACCGCGA CGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGC TCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATG TCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGC ACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAGTTTAGCG AGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGC CTGAAACCGAACTGCCCGCTGTTCTACAACCGGTCGCGGAGGCTATGGATGCGATC GCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAAT CGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTA TCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGA TGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGG ATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACT GGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGA GGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCG GAGCTTGCAGGATCGCCACGACTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAA CTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGA TGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCG CAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAA ACCGACGCCCCAGCACTCGTCCGAGGGCAAAGAAATAGAGTAGATGCCGACCGGAT CTGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTGTGAGTAGTTCCCAGATAA GGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGAGCATATAAGAAACCCTTA GTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCA AAATCCAGTACTAAAATCCAGATC.

Split-Marker Cassettes Methods

[229] In some embodiments, a gene described herein is replaced with a hygromycin resistance gene. In some embodiments, a PsiD gene is replaced with a hygromycin resistance gene. In some embodiments, a PsiH gene is replaced with a hygromycin resistance gene. In some embodiments, a PsiH2 gene is replaced with a hygromycin resistance gene. In some embodiments, a PsiK gene is replaced with a hygromycin resistance gene. In some embodiments, a PsiL gene is replaced with a hygromycin resistance gene. In some embodiments, a PsiM gene is replaced with a hygromycin resistance gene. In some embodiments, a PsiP gene is replaced with a hygromycin resistance gene. In some embodiments, a PsiP2 gene is replaced with a hygromycin resistance gene. In some embodiments, a PsiTl gene is replaced with a hygromycin resistance gene. In some embodiments, a PsiR gene is replaced with a hygromycin resistance gene. In some embodiments, a PsiT2 gene is replaced with a hygromycin resistance gene. In some embodiments the hygromycin resistance gene is 35s hygromycin. In some embodiments, one or more cassettes are comprised in a plasmid described herein. In some embodiments the cassette is a cassette described in TABLE 8B. In some embodiments, the DNA component of a gene described herein is split into two cassettes. In some embodiments, these can be referred to as split marker cassettes. In some embodiments, the split marker cassettes are used in conjunction with in vitro assembled Cas9-guide RNA ribonucleoproteins (TABLE 8B). In some embodiments the split marker cassettes are used in conjunction with in vitro assembled Cas9-guide RNA ribonucleoproteins for rapid and efficient gene deletions. In some embodiments the cassette is at least 1 kb. In some embodiments the cassette is at least 1 kb, at least 1.1 kb, at least 1.2 kb, at least 1.3 kb, at least 1.4 kb, at least 1.5 kb, at least 1.6 kb, at least 1.7 kb, at least 1.8 kb, at least 1.9 kb, or at least 2.0 kb. In some embodiments, multiple guide RNAs are used. In some embodiments a first guide RNA is located at the start of a gene described herein, and a second guide RNA is placed at the end of the same gene described herein for replacement. In some embodiments, the guide RNAs are each independently selected from a guide RNA sequence in TABLE 8A. In some embodiments the split marker cassette has a sequence listed in TABLE 8B. In some embodiments, an upstream homology arm sequence (UHA) can be at least 500 base pairs (bp), at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least 850 bp, at least 900 bp, at least 950 bp, or at least lOOObp. In some embodiments, a downstream homology arm sequence (DHA) can be at least 500 base pairs (bp), at least 550 bp, at least 600 bp, at least 650 bp, at least 700 bp, at least 750 bp, at least 800 bp, at least 850 bp, at least 900 bp, at least 950 bp, or at least lOOObp.

[230] TABLE 8A. HDR guide RNA sequences

TABLE 8B. Exemplary Cassettes for HDR integration of PsiD

[231] In some embodiments, HDR methods include using sequence to replace a promoter of a gene described herein with a GPDi promoter. In some embodiments, the GPDi promoter has a sequence comprising: GAGGTCCGCAAGTAGATTGAAAGTTCAGTACGTTTTTAACAATAGAGCATTTTCGAG GCTTGCGTCATTCTGTGTCAGGCTAGCAGTTTATAAGCGTTGAGGATCTAGAGCTGC TGTTCCCGCGTCTCGAATGTTCTCGGTGTTTAGGGGT.

[232] TAGCAATCTGATATGATAATAATTTGTGATGACATCGATAGTACAAAAACCC CAATTCCGGTCACATCCACCATCTCCGTTTTCTCCCATCTACACACAACAAGCTCATC GCCGTTTGTCTCTCGCTTGCATACCACCCAGCAGCTCACTGATG

[233] TCGACTTGTAG In some embodiments a repair template is used with homology arms on either side of the GPDi promoter. In some embodiments, the homology arms are 111 base pairs to the left of the GPDi promoter. In some embodiments, the homology arms are 125 base pairs to the right of the GPDi promoter. In some embodiments, the homology arms are 111 base pairs to the left of the GPDi promoter and the homology arms are 125 base pairs to the right of the GPDi promoter. In embodiments describing directionality, interpretation should be 5’ to 3’ directionality unless otherwise indicated. In some embodiments, the first (left) HDR guide is: CTCCCAACACTTGATCATGC. In some embodiments, the second (right) HDR guide is: TCACCTGCATGATCAAGTGT. In some embodiments the repair template sequence for HDR methods performed on a genetically modified organism is: CGAGCATCTCTCTCTAGTCATAGTTTATCTTTGTATAAATGGGGGCCTCAACGCAAG GCCGCAAAACTACTCCCAACTTTTATAACTCATTTCTGCTCCCAACACTTGATCGAG GTCCGCAAGTAGATTGAAAGTTCAGTACGTTTTTAACAATAGAGCATTTTCGAGGCT TGCGTCATTCTGTGTCAGGCTAGCAGTTTATAAGCGTTGAGGATCTAGAGCTGCTGT TCCCGCGTCTCGAATGTTCTCGGTGTTTAGGGGTTAGCAATCTGATATGATAATAAT TTGTGATGACATCGATAGTACAAAAACCCCAATTCCGGTCACATCCACCATCTCCGT TTTCTCCCATCTACACACAACAAGCTCATCGCCGTTTGTCTCTCGCTTGCATACCACC CAGCAGCTCACTGATGTCGACTTGTAGATGCAGGTGATACCCGCGTGCAACTCGGCG TACGTCGTTTTTATTCGCTGACTTCACCCGCTAATTACTATAACTTGAAAACACAGA GCAATAAGATCACTATGTCCTACTCCCGAGTCTTTGAG.

[234] This disclosure provides methods of genetically modifying organism for the production of one or more alkaloids. The genetic modification can be accomplished using a genome editing (also called gene editing) system refers to a group of technologies that give the ability to change an organism's DNA. Compositions and methods described herein take advantage of genome editing systems to make targeted edits in an organism’s genome and thereby produce one or more alkaloids that are of interest. To that end, the genome editing systems as used herein can possess programmable nucleases. In some embodiments, the genome editing system comprises a zinc-finger nuclease (ZFN). A zinc finger nuclease is an artificial endonuclease that can comprise a designed zinc finger protein (ZFP) fused to a cleavage domain, such as, a FokI restriction enzyme. In some embodiments, the genome editing system comprises a transcription activator-like effector nuclease (TALEN). TALENs are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA- binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. In some embodiments, the genome editing system is a meganuclease. In some embodiments, a gene editing system is used in incorporate an exogenous nucleic acid into a fungal, wherein incorporation of the exogenous nucleic acid results in a genetic modification that modulates production of an alkaloid. In some embodiments, the exogenous nucleic acid comprises a sequence that is at least about: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or is 100% identical to one of the sequences listed in TABLE 2. In some embodiments, the gene editing system is a CRISPR system, such as the CRISPR-Cas9 endonuclease system. In some embodiments, the gene editing system is a CRISPR system, such as the CRISPR-spCas9 endonuclease system.

[235] In some embodiments, the exogenous nucleic acid includes a promoter sequence. Increasing expression of designed gene products may be achieved by synthetically increasing expression by modulating promoter regions or inserting stronger promoters upstream of desired gene sequences. In some embodiments, for example, a gene promoter such as 35S gene promoter can be used.

[236] In some embodiments, the exogenous nucleic acid can include a barcode or watermark sequence, which may be referred to as “a barcode”. A barcode can comprise a non-natural sequence. In some embodiments, the barcode can be used to identify transgenic organisms via genotyping. In some embodiments, the exogenous nucleic acid can include a selectable marker, such as an antibiotic resistance gene. Selectable marker genes, also referred to herein as “selection markers,” include, for example, a hygromycin resistance gene.

[237] In some embodiments, a unique sequence is embedded into the genome of a genetically modified organism described herein using CRISPR methods for identification purposes. In some embodiments, this is referred to as a marker or marker sequence. In some embodiments this is referred to as a watermark sequence. In some embodiments, this is referred to as an intergenic sequence, or a portion thereof. In some embodiments, this is referred to as an intergenic watermark sequence. In some cases, the exogenous nucleic acid can include a barcode. In some embodiments, this is referred to as barcoding. A barcode can comprise a non-natural sequence. In some embodiments, the barcode can be used to identify transgenic organisms via genotyping. In some embodiments, the exogenous nucleic acid can include a selectable marker, such as an antibiotic resistance gene. Selectable marker genes can include, for example, a hygromycin resistance gene. In some embodiments, this can be accomplished using resistance vector gene sequences like those shown in Table 5A.

[238] This disclosure provides methods of genetically modifying organism for the production of one or more alkaloids. The genetic modification can be accomplished using a genome editing (also called gene editing) system refers to a group of technologies that give the ability to change an organism's DNA. Compositions and methods described herein take advantage of genome editing systems to make targeted edits in an organism’s genome and thereby produce one or more alkaloids that are of interest. To that end, the genome editing systems as used herein can possess programmable nucleases. In some embodiments, the genome editing system comprises a zinc-finger nuclease (ZFN). A zinc finger nuclease is an artificial endonuclease that can comprise a designed zinc finger protein (ZFP) fused to a cleavage domain, such as, a FokI restriction enzyme. In some embodiments, the genome editing system comprises a transcription activator-like effector nuclease (TALEN). TALENs are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA- binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. In some embodiments, the genome editing system is a meganuclease. In some embodiments, a gene editing system is used in incorporate an exogenous nucleic acid into a fungal, wherein incorporation of the exogenous nucleic acid results in a genetic modification that modulates production of an alkaloid. In some embodiments, the exogenous nucleic acid comprises a sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or is 100% identical to one of the sequences listed in TABLE 2, or TABLE 3A-3B. In some embodiments, the gene editing system is a CRISPR system, such as the CRISPR-Cas9 endonuclease system.

[239] In some cases, an endonuclease or a nuclease or a polypeptide encoding a nuclease can be selected from the group consisting of Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Cpfl, c2cl, c2c3, Cas9HiFi, homologues thereof or modified versions thereof. A Cas protein can be Cas9. A Cas9 can create a double strand break in said at least one endogenous genome. In some cases, an endonuclease or a nuclease or a polypeptide encoding a nuclease can be Cas9 or a polypeptide encoding Cas9. In some cases, an endonuclease or a nuclease or a polypeptide encoding a nuclease can be catalytically dead. In some cases, an endonuclease or a nuclease or a polypeptide encoding a nuclease can be a catalytically dead Cas9 or a polypeptide encoding a catalytically dead Cas9. The Cas endonuclease can be optimized for expression in a fungal cell. In some embodiments, the Cas endonuclease is codon optimized. Codon optimization is a process used to improve gene expression and increase the translational efficiency of a gene of interest by accommodating codon bias of organism to be modified. In some embodiments, the Cas endonuclease comprises a sequence that is at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identical to: gataaaaaatattcaatcggattggatatcggaacaaactcagtcggatgggcagtcatcacagatgaatataaagtcccatcaaaaaaattc aaagtcttgggaaacacagatagacattcaatcaaaaaaaacttgatcggagcattgttgttcgattcaggagaaacagcagaagcaacaag attgaaaagaacagcaagaagaagatatacaagaagaaaaaacagaatctgctatttgcaagaaatcttctcaaacgaaatggcaaaagtc gatgattcattcttccatagattggaagaatcattcttggtcgaagaagataaaaaacatgaaagacatccaatcttcggaaacatcgtcgatg aagtcgcatatcatgaaaaatatccaacaatctatcatttgagaaaaaaattggtcgattcaacagataaagcagatttgagattgatctatttgg cattggcacatatgatcaaattcagaggacatttcttgatcgaaggagatttgaacccagataactcagatgtcgataaattgttcatccaattg gtccaaacatataaccaattgttcgaagaaaacccaatcaacgcatcaggagtcgatgcaaaagcaatcttgtcagcaagattgtcaaaatca agaagattggaaaacttgatcgcacaattgccaggagaaaaaaaaaacggattgttcggaaacttgatcgcattgtcattgggattgacacc aaacttcaaatcaaacttcgatttggcagaagatgcaaaattgcaattgtcaaaagatacatatgatgatgatttggataacttgttggcacaaat cggagatcaatatgcagatttgttcttggcagcaaaaaacttgtcagatgcaatcttgttgtcagatatcttgagagtcaacacagaaatcacaa aagcaccattgtcagcatcaatgatcaaaagatatgatgaacatcatcaagatttgacattgttgaaagcattggtcagacaacaattgccaga aaaatataaagaaatcttcttcgatcaatcaaaaaacggatatgcaggatatatcgatggaggagcatcacaagaagaattctataaattcatc aaaccaatcttggaaaaaatggatggaacagaagaattgttggtcaaattgaacagagaagatttgttgagaaaacaaagaacattcgataa cggatcaatcccacatcaaatccatttgggagaattgcatgcaatcttgagaagacaagaagatttctatccattcttgaaagataacagagaa aaaatcgaaaaaatcttgacattcagaatcccatattatgtcggaccattggcaagaggaaactcaagattcgcatggatgacaagaaaatca gaagaaacaatcacaccatggaacttcgaagaagtcgtcgataaaggagcatcagcacaatcattcatcgaaagaatgacaaacttcgata aaaacttgccaaacgaaaaagtcttgccaaaacattcattgttgtatgaatatttcacagtctataacgaattgacaaaagtcaaatatgtcaca gaaggaatgagaaaaccagcattcttgtcaggagaacaaaaaaaagcaatcgtcgatttgttgttcaaaacaaacagaaaagtcacagtca aacaattgaaagaagattatttcaaaaaaatcgaatgcttcgattcagtcgaaatctcaggagtcgaagatagattcaacgcatcattgggaac atatcatgatttgttgaaaatcatcaaagataaagatttcttggataacgaagaaaacgaagatatcttggaagatatcgtcttgacattgacatt gttcgaagatagagaaatgatcgaagaaagattgaaaacatatgcacatttgttcgatgataaagtcatgaaacaattgaaaagaagaagata tacaggatggggaagattgtcaagaaaattgatcaacggaatcagagataaacaatcaggaaaaacaatcttggatttcttgaaatcagatg gattcgcaaacagaaacttcatgcaattgatccatgatgattcattgacattcaaagaagatatccaaaaagcacaagtctcaggacaaggag attcattgcatgaacatatcgcaaacttggcaggatcaccagcaatcaaaaaaggaatcttgcaaacagtcaaagtcgtcgatgaattggtca aagtcatgggaagacataaaccagaaaacatcgtcatcgaaatggcaagagaaaaccaaacaacacaaaaaggacaaaaaaactcaag agaaagaatgaaaagaatcgaagaaggaatcaaagaattgggatcacaaatcttgaaagaacatccagtcgaaaacacacaattgcaaaa cgaaaaattgtatttgtattatttgcaaaacggaagagatatgtatgtcgatcaagaattggatatcaacagattgtcagattatgatgtcgatcat atcgtcccacaatcattcttgaaagatgattcaatcgataacaaagtcttgacaagatcagataaaaacagaggaaaatcagataacgtccca tcagaagaagtcgtcaaaaaaatgaaaaactattggagacaattgttgaacgcaaaattgatcacacaaagaaaattcgataacttgacaaaa gcagaaagaggaggattgtcagaattggataaagcaggattcatcaaaagacaattggtcgaaacaagacaaatcacaaaacatgtcgca caaatcttggattcaagaatgaacacaaaatatgatgaaaacgataaattgatcagagaagtcaaagtcatcacattgaaatcaaaattggttt cagatttcagaaaagatttccaattctataaagtcagagaaatcaacaactatcatcatgcacatgatgcatatttgaacgcagtcgtcggaac agcattgatcaaaaaatatccaaaattggaatcagaattcgtctatggagattataaagtctatgatgtcagaaaaatgatcgcaaaatcagaa caagaaatcggaaaagcaacagcaaaatatttcttctattcaaacatcatgaacttcttcaaaacagaaatcacattggcaaacggagaaatc agaaaaagaccattgatcgaaacaaacggagaaacaggagaaatcgtctgggataaaggaagagatttcgcaacagtcagaaaagtcttg tcaatgccacaagtcaacatcgtcaaaaaaacagaagtccaaacaggaggattctcaaaagaatcaatcttgccaaaaagaaactcagata aattgatcgcaagaaaaaaagattgggatccaaaaaaatatggaggattcgattcaccaacagtcgcatattcagtcttggtcgtcgcaaaag tcgaaaaaggaaaatcaaaaaaattgaaatcagtcaaagaattgttgggaatcacaatcatggaaagatcatcattcgaaaaaaacccaatc gatttcttggaagcaaaaggatataaagaagtcaaaaaagatttgatcatcaaattgccaaaatattcattgttcgaattggaaaacggaagaa aaagaatgttggcatcagcaggagaattgcaaaaaggaaacgaattggcattgccatcaaaatatgtcaacttcttgtatttggcatcacattat gaaaaattgaaaggatcaccagaagataacgaacaaaaacaattgttcgtcgaacaacataaacattatttggatgaaatcatcgaacaaatc tcagaattctcaaaaagagtcatcttggcagatgcaaacttggataaagtcttgtcagcatataacaaacatagagataaaccaatcagagaa caagcagaaaacatcatccatttgttcacattgacaaacttgggagcaccagcagcattcaaatatttcgatacaacaatcgatagaaaaaga tatacatcaacaaaagaagtcttggatgcaacattgatccatcaatcaatcacaggattgtatgaaacaagaatcgatttgtcacaattgggag gagat (SEQ ID NO: 202).

[240] In some embodiments, the Cas endonuclease comprises a nuclear localization signal.

The nuclear localization signal can comprise an optimized sequence that is at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identical to ccaaaaaaaaaaagaaaagtcggaatccatggagtcccagcagca (SEQ ID NO: 201). The nuclear localization signal can be connected to the Cas endonuclease via a linker. The linker can be codon optimized and comprise a sequence that is at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identical to ggaatccatggagtcccagcagcaccaaaaaaaaaaagaaaagtctga (SEQ ID NO: 203).

[241] In some embodiments, the Cas endonuclease comprises a FLAG tag. The FLAG tag comprises an artificial antigen to which specific, high affinity monoclonal antibodies have been developed and hence can be used for protein purification by affinity chromatography and also can be used for locating proteins within living cells. The FLAG tag may be attached by a codon optimized linker that is least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identical to gattataaagatcatgatggagattataaagatcatgatatcgattataaagatgatgatgataaagcagca SEQ ID NO: 200.

[242] In some cases, a CRISPR enzyme coupled with a guide polynucleotide can be delivered into a genetically modified organism by a vector comprising a nucleic acid encoding the CRISPR enzyme and the guide polynucleotide. In an aspect, a vector can be a binary vector or a Ti plasmid. In an aspect, a vector further comprises a selection marker or a reporter gene. In some cases, a RNP, complex, or vector can be delivered via electroporation, microinjection, mechanical cell deformation, lipid nanoparticles, AAV, lentivirus, agrobacterium mediated transformation, biolistic particle bombardment, or protoplast transformation. In some cases, a RNP, mRNA, or vector further comprises a donor polynucleotide or a nucleic acid encoding the donor polynucleotide. In an aspect, a donor polynucleotide comprises homology to sequences flanking a target sequence. In an aspect, a donor polynucleotide further comprises a barcode, a reporter gene, or a selection marker.

[243] In some embodiments, the Cas endonuclease is employed as a base editor. In some embodiments, the Cas nuclease is part of a Cas system. In some embodiments, the Cas endonuclease is a part of a fusion protein. In some embodiments, the fusion protein introduces nucleobase editing into a sequence described herein. In some embodiments, the base edit results in a specific alteration in the sequence encoding a protein of interest. In some embodiments, the base edit results in one or more specific alterations in the sequence encoding a protein of interest. In some embodiments, the Cas system comprises an adenine base editor. In some embodiments, the Cas system comprises a cytosine base editor. In some embodiments, the Cas system comprises a cytosine-to-guanine base editor. In some embodiments, base editing results in one point mutation to a sequence described, herein. In some embodiments, base editing results in more than one point mutation to a sequence described, herein, and the endonuclease is coupled to a reverse-transcriptase enzyme. In some embodiments, a prime editing Cas system further comprises a prime-editing guide RNA (pegRNA). PegRNA targets editing machinery at a specific site on a genome, and additionally contains a template sequence and a primer-binding sequence. The template sequence encodes the intended genome-sequence change.

[244] In some embodiments, the method of introducing a genetic modification includes prime editing methods. In prime editing, an endonuclease makes a single-stranded cut in the target sequence.

[245] In some embodiments, base editing or prime editing result in the alteration of genomic sequences. In some embodiments, base editing or prime editing result in the alteration of genomic sequences that control gene expression. In some embodiments, base editing or prime editing result in the increased gene expression of a gene of interest. In some embodiments, base editing or prime editing result in the decreased gene expression of a gene of interest.

[246] In some embodiments, genetically modifying an organism, as described herein, is achieved by introducing an exogenous nucleic acid, e.g., a donor sequence, into a cell of the organism, for example, fungal cell, e.g., a fungal protoplast. The exogenous nucleic acid may encode one or more gene products that, when expressed by the genetically modified organism, result in the genetically modified organism producing an increased amount of the one or more alkaloids as compared to a comparable wild-type organism. In some embodiments, the one or more genes can be one of the genes listed in Table 1 or Table 2. In some embodiments, the one or more genes can be one of the genes listed in TABLE 1, TABLE 2, or TABLE 3A-3B. In some embodiments, one or more copies of the one or more genes included in Table 1 or Table 2 are provided by the exogenous nucleic acid. For example, in some instances at least 1, 2, 3, 4, 5, 6, or 7 copies of the one or more genes are introduced into the genetically modified organism with the exogenous nucleic acid. In some embodiments, the genetic modification results in the genetically modified organism expressing one or more of the polynucleotides listed in TABLE 1, TABLE 2, TABLE 3A-TABLE 3B. In some embodiments, at least a portion of the exogenous nucleic acid can be integrated into the genome of the organism. For example, the exogenous nucleic acid can be inserted into a genomic break. In some embodiments, at least a portion of the exogenous nucleic acid includes sequences that are homologous to sequences flanking a target sequence for targeted integration.

[247] In some embodiments, the exogenous nucleic acid includes a promoter sequence. Increasing expression of a designed gene products may be achieved by synthetically increasing expression by modulating promoter regions or inserting stronger promoters upstream of desired gene sequences. In some embodiments, for example, a gene promoter such as 35S gene promoter are used.

[248] In some embodiments, the exogenous nucleic acid can include a barcode. The unique sequence is embedded into the genome of a genetically modified organism described herein using CRISPR methods for identification purposes. In some embodiments, this is referred to as a marker or marker sequence. In some embodiments this is referred to as a watermark sequence. In some embodiments, this is referred to as an intergenic sequence, or a portion thereof. In some embodiments, this is referred to as an intergenic watermark sequence. In some embodiments, this is referred to as barcoding. A barcode can comprise a non-natural sequence. In some embodiments, the barcode can be used to identify transgenic organisms via genotyping. In some embodiments, the exogenous nucleic acid can include a selectable marker, such as an antibiotic resistance gene. Selectable marker genes can include, for example, a hygromycin resistance gene.

[249] In some embodiments, a unique sequence is embedded into the genome of a genetically modified organism described herein using CRISPR methods for identification purposes. In some embodiments, this is referred to as a marker or marker sequence. In some embodiments this is referred to as a watermark sequence. In some embodiments, this is referred to as an intergenic sequence, or a portion thereof. In some embodiments, this is referred to as an intergenic watermark sequence. In some embodiments, this is referred to as barcoding. In some cases, the exogenous nucleic acid can include a barcode. The unique sequence is embedded into the genome of a genetically modified organism described herein using CRISPR methods for identification purposes. In some embodiments, this is referred to as a marker or marker sequence. In some embodiments this is referred to as a watermark sequence. In some embodiments, this is referred to as an intergenic sequence, or a portion thereof. In some embodiments, this is referred to as an intergenic watermark sequence. In some embodiments, this is referred to as barcoding. A barcode can comprise a non-natural sequence. In some embodiments, the barcode can be used to identify transgenic organisms via genotyping. In some embodiments, the exogenous nucleic acid can include a selectable marker, such as an antibiotic resistance gene. Selectable marker genes can include, for example, a hygromycin resistance gene. In some embodiments, this can be accomplished using resistance vector gene sequences like those shown in TABLE 5 A.

[250] In some embodiments, the hygromycin resistance gene sequence is SEQ ID NO. 302. In some embodiments, the sequence encoding the marker can be incorporated into the genetically modified cell or organism, for instance a fungal cell, yeast cell or plant cell as described herein. In some cases, a marker serves as a selection or screening device may function in a regenerable genetically modified organism to produce a compound that would confer upon a tissue in said organism resistance to an otherwise toxic compound. In some embodiments, the incorporated sequence encoding the marker may by subsequently removed from the transformed genome. Removal of a sequence encoding a marker may be facilitated by the presence of direct repeats before and after the region encoding the marker. In some embodiments, the marker sequence is followed by a protospacer adjacent motif (PAM), in order to provide appropriate cleavage by a Cas nuclease. [251] In some embodiments, the exogenous nucleic acid can be introduced into the genetically modified organism by transformation or transfection. The exogenous nucleic acid can be introduced before, after, or concurrently with a gene editing system.

[252] In some embodiments, a hygromycin resistance gene is used. In some embodiments, the sequence encoding the marker can be incorporated into the genetically modified cell or organism, for instance a fungal cell, yeast cell or plant cell as described herein. In some cases, a marker serves as a selection or screening device may function in a regenerable genetically modified organism to produce a compound that would confer upon a tissue in said organism resistance to an otherwise toxic compound. In some embodiments, the incorporated sequence encoding the marker may by subsequently removed from the transformed genome. Removal of a sequence encoding a marker may be facilitated by the presence of direct repeats before and after the region encoding the marker. In some embodiments, the marker sequence is followed by a protospacer adjacent motif (PAM), in order to provide appropriate cleavage by a Cas nuclease.

[253] In some instances, the exogenous nucleic acid can be introduced into the genetically modified organism by transformation or transfection. Transformation appropriate transformation techniques can include but are not limited to: electroporation of fungi protoplasts; liposome- mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of ceils; micro-projectile bombardment of cells; vacuum infiltration; and Agrobacterium tumeficiens mediated transformation. Transformation can mean introducing a nucleotide sequence into a cell in a manner to cause stable or transient expression of the sequence.

[254] Following transformation, fungi or other organisms can be selected using a dominant selectable marker incorporated into, for example, the transformation vector. In certain embodiments, such marker confers antibiotic or herbicide resistance on the transformed fungi or other organisms, and selection of transformants can be accomplished by exposing the fungi and other organisms to appropriate concentrations of the antibiotic or herbicide. After transformed fungi or other organisms are selected and grown to maturity, those fungi and other organisms showing a modified trait are identified. The modified trait can be any of those traits described above. Additionally, expression levels or activity of the polypeptide or polynucleotide of the disclosure can be determined by analyzing mRNA expression, using Northern blots, RT-PCR, RNA seq or microarrays, or protein expression using immunoblots or Western blots or gel shift assays.

[255] Suitable methods for transformation of fungal or other cells for use with the current disclosure are believed to include virtually any method by which a nucleic acid can be introduced into a cell, such as by direct delivery of DNA such as by PEG-mediated transformation of protoplasts, by desiccation/inhibition-mediated DNA uptake, by electroporation, by agitation with silicon carbide fibers, by Agrobacterium-mediated transformation and by acceleration of DNA coated particles. Through the application of techniques such as these, the cells of virtually any fugus species may be stably transformed, and these cells developed into transgenic fungi.

Gene Editing Systems

[256] Agrobacterium-mediated transfer can be used to introduce an exogenous nucleic acid into an organism selected for genetic modification, such as a fungal cell. In some instances, the exogenous nucleic acid can be introduced into whole fungal tissues, thereby by passing the need for regeneration of an intact fungus from a protoplast. The use of agrobacterium-mediated transformation can be used to integrate one or more vectors into the genetically modified organisms, including vectors or sequences encoding gene-editing systems, such as CRISPR systems or donor sequences.

[257] This disclosure includes advances in vectors for agrobacterium-mediated gene transfer by providing improved the arrangement of genes and restriction on sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. In some embodiments, a vector can have convenient multi-linker regions flanked by a promoter and a poly adenylation site for direct expression of inserted polypeptide coding genes and are suitable for purposes described herein. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations.

[258] In some embodiments, aspects, a fungal cell, yeast cell, plant cell, may be modified using electroporation. To effect transformation by electroporation, one may employ either friable tissues, such as a suspension culture of cells or embryogenic callus or alternatively one may transform immature embryos or other organized tissue directly. In some cases, electroporation may comprise 2 pulses, 3 pulses, 4 pulses, 5 pulses 6 pulses, 7 pulses, 8 pulses, 9 pulses, or 10 or more pulses. In some embodiments, protoplasts of fungi and/or plants may be used for electroporation transformation.

[259] Another method for delivering transforming DNA segments to fungal cells and cells derived from other organisms in accordance with the disclosure is microprojectile bombardment. In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary' particles include those comprised of tungsten, platinum, and preferably, gold. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary' for DNA delivery' to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. In some embodiments, DNA-coated particles may increase the level of DNA delivery via particle bombardment. For the bombardment, cells in suspension are concentrated on filters or solid culture medium. Alternatively, immature embryos or other target cells may be arranged on solid culture medium. The cells that can be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate. In some cases, a starting cell density for genomic editing may be varied to optimize editing efficiency and/or cell viability.

[260] In some embodiments, fungi, yeast or plants of the present disclosure can be used to produce new plant varieties. In some embodiments, the plants are used to develop new, unique and superior varieties or hybrids with desired phenotypes. In some embodiments, selection methods, e.g., molecular marker assisted selection, can be combined with breeding methods to accelerate the process. In some embodiments, a method comprises (i) crossing any organism provided herein comprising the expression cassette as a donor to a recipient organism line to create a FI population, (ii) selecting offspring that have expression cassette. Optionally, the offspring can be further selected by testing the expression of the gene of interest. In some embodiments, complete chromosomes of a donor organism are transferred. For example, the transgenic organism with an expression cassette can serve as a male or female parent in a cross pollination to produce offspring by receiving a transgene from a donor thereby generating offspring having an expression cassette. In a method for producing organisms having the expression cassette, protoplast fusion can also be used for the transfer of the transgene from a donor to a recipient. Protoplast fusion is an induced or spontaneous union, such as a somatic hybridization, between two or more protoplasts (cells in which the cell walls are removed by enzymatic treatment) to produce a single bi- or multi -nucleate cell. In another aspect, mass selection can be utilized. In mass selection, desirable individual plants are chosen, harvested, and the seed composited without progeny testing to produce the following generation. Since selection is based on the maternal parent only, and there is no control over pollination, mass selection amounts to a form of random mating with selection. As stated herein, the purpose of mass selection is to increase the proportion of superior genotypes m the population.

[261] This disclosure provides gene editing systems for genetically modifying organisms. The gene editing system can be selected form the group consisting of a CRISPR system, TALEN, Zinc Finger, transposon-based, ZEN, meganuclease, Mega-TAL, and any combination thereof. In some embodiments, the gene editing system is directed to a target of interest by a guide polynucleotide. In some embodiments, the gene editing system involves an endonuclease or a nuclease or a polypeptide encoding a nuclease can be from a CRISPR (clustered regularly interspaced short palindromic repeats) system. An endonuclease or a nuclease or a polypeptide encoding a nuclease can be a Cas or a polypeptide encoding a Cas. [262] CRISPR can refer to a family of DNA repeats found in certain bacterial genomes. In some instances, the CRISPR protein can include a Cas9 endonuclease, which can form a complex with a crRNA/tracrRNA hybrid to recognize, bind, and ultimately cleave foreign DNA at a target site. The crRNA/tracrRNA hybrid can be designed as a single guide RNA (gRNA). For example, any one or more of the guide RNAs shown in TABLE 9, through TABLE 16. In some embodiments, the gRNA sequence is followed by a protospacer adjacent motif (PAM), in order to provide appropriate cleavage by a Cas nuclease.

[263] The recognition of a target DNA target region can depend on a protospacer adjacent motif (PAM) which can be located at the 3 '-terminus of a 20 bp target sequence. Once the CRISPR complex (e.g., Cas9 and associated guide RNA) recognizes the target DNA sequence, the CRISPR complex can generate a double strand break (DSB) at the DNA target locus. In some instances, one of two cellular DNA repair mechanisms, non-homologous end joining (NHEJ) and homologous recombination (HR), can play a role in precise genome editing and gene manipulation. For example, NHEJ, which is sometimes regarded as an error-prone repair mechanism that generates either short insertions or deletions of nucleotides in close proximity to the DSB site(s), can be used. If these short insertions or deletions exist in a gene coding region, or within a portion of the promoter involved in recruiting proteins involved in transcription, the function of the endogenous gene, for example a gene encoding psilocybin phosphatase, can be disrupted. Consequently, this procedure can be used for generating gene mutations. In other embodiments, a homology independent targeted integration (HITI) strategy can be used which allows fragments (e.g., exogenous nucleic acids) to be integrated into the genome by NHEJ repair.

[264] Various versions of CRISPR systems can be used. In some instances, the CRISPR system can be introduced into the genome of a target organism using Agrobacterium tumefaciens- mediated transformation. When the expression of Cas protein and guide RNA can be under the control of either a constitutive or inducible promoter. For example, in some embodiments, the Cas protein is under the control of a GDP gene protomer, while the guide RNA is under the control of a U6 gene promoter. In some embodiments, the guide RNA is inserted directly downstream of a P. cubensis U6 promoter and directly upstream of the guide RNA scaffold sequence. In some instances, the Cas protein is optimized for use in a fungal cell.

[265] In some cases, a Cas protein comprises Cast, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, CasSt, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, CseSe, Csci, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, C2cl, C2c2, C2c3, Cpfl, Cas9HiFi, CARF, DinG, homologues thereof, or modified versions thereof. In some cases, a Cas protein can be a Cas9. In some cases, Cas9 is a modified Cas9 that binds to a canonical PAM. In some cases, Cas9 recognizes a non-canonical PAM. In some cases, a guide polynucleotide binds a target sequence 3-10 nucleotides from a PAM. In some cases, a CRISPR enzyme coupled with a guide polynucleotide can be delivered into a genetically modified organism as an RNP. In some cases, a CRISPR enzyme coupled with a guide polynucleotide can be delivered into a genetically modified organism by a mRNA encoding the CRISPR enzyme and the guide polynucleotide. In some cases, the Cas protein is referred to as a Cas endonuclease, or endonuclease. In some cases, an endonuclease or a nuclease or a polypeptide encoding a nuclease can be Cas9 or a polypeptide encoding Cas9. In some cases, an endonuclease or a nuclease or a polypeptide encoding a nuclease can be catalytically dead. In some cases, an endonuclease or a nuclease or a polypeptide encoding a nuclease can be a catalytically dead Cas9 or a polypeptide encoding a catalytically dead Cas9.

[266] The Cas endonuclease can be optimized for expression in a fungal cell. In some embodiments, the Cas endonuclease is codon optimized. Codon optimization is a process used to improve gene expression and increase the translational efficiency of a gene of interest by accommodating codon bias of organism to be modified. In some embodiments, the Cas endonuclease comprises a sequence that is at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identical to SEQ ID NO: 203. In some embodiments, the Cas endonuclease comprises a nuclear localization signal. The nuclear localization signal can comprise a sequence that is at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identical to SEQ ID NO: 201. In some embodiments, the Cas endonuclease comprises a FLAG tag. The FLAG tag comprises an artificial antigen to which specific, high affinity monoclonal antibodies have been developed and hence can be used for protein purification by affinity chromatography and also can be used for locating proteins within living cells. The FLAG tag may be attached by a codon optimized linker that is least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identical to SEQ ID NO: 200.

[267] In some cases, a CRISPR enzyme coupled with a guide polynucleotide can be delivered into a genetically modified organism by a vector comprising a nucleic acid encoding the CRISPR enzyme and the guide polynucleotide. In an aspect, a vector can be a binary vector or a Ti plasmid. In an aspect, a vector further comprises a selection marker or a reporter gene. In some cases, a RNP, complex, or vector can be delivered via electroporation, microinjection, mechanical cell deformation, lipid nanoparticles, AAV, lentivirus, agrobacterium mediated transformation, biolistic particle bombardment, or protoplast transformation. In some cases, a RNP, mRNA, or vector further comprises a donor polynucleotide or a nucleic acid encoding the donor polynucleotide. In an aspect, a donor polynucleotide comprises homology to sequences flanking a target sequence. In an aspect, a donor polynucleotide further comprises a barcode, a reporter gene, or a selection marker.

[268] In some embodiments, the Cas endonuclease is employed as a base editor. In some embodiments, the Cas nuclease is part of a Cas system. In some embodiments, the Cas endonuclease is a part of a fusion protein. In some embodiments, the fusion protein introduces nucleobase editing into a sequence described herein. In some embodiments, the base edit results in a specific alteration in the sequence encoding a protein of interest. In some embodiments, the base edit results in one or more specific alterations in the sequence encoding a protein of interest. In some embodiments, the Cas system comprises an adenine base editor. In some embodiments, the Cas system comprises a cytosine base editor. In some embodiments, the Cas system comprises a cytosine-to-guanine base editor. In some embodiments, base editing results in one point mutation to a sequence described, herein. In some embodiments, base editing results in more than one point mutation to a sequence described, herein, and the endonuclease is coupled to a reversetranscriptase enzyme. In some embodiments, a prime editing Cas system further comprises a prime-editing guide RNA (pegRNA). PegRNA targets editing machinery at a specific site on a genome, and additionally contains a template sequence and a primer-binding sequence. The template sequence encodes the intended genome-sequence change.

[269] In some embodiments, the method of introducing a genetic modification includes prime editing methods. In prime editing, an endonuclease makes a single-stranded cut in the target sequence.

[270] In some embodiments, base editing or prime editing result in the alteration of genomic sequences. In some embodiments, base editing or prime editing result in the alteration of genomic sequences that control gene expression. In some embodiments, base editing or prime editing result in the increased gene expression of a gene of interest. In some embodiments, base editing or prime editing result in the decreased gene expression of a gene of interest.

[271] In some embodiments, the gene editing system further comprises an exogenous nucleic acid. In some embodiments, the exogenous nucleic acid comprises a psilocybin synthase gene. In some embodiments, exogenous nucleic acid comprises any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin- related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop- helix transcription factor that binds to an E-box motif. In some embodiments, the exogenous nucleic acid comprises a sequence that has at least a 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identity to one of SEQ ID NOS: 1-16, 67-70, or 90-96. In some embodiments, the gene editing system is used to integrate the exogenous nucleic acid into a psilocybin synthase gene. In some embodiments, the gene editing system is used to add or delete one or more nucleic acids of a psilocybin synthase gene, e.g., one of the genes listed in TABLE 2, thereby creating a frameshift mutation that results in the downregulation of the gene. In some embodiments, expression of the gene is reduced by about 50 percent, 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent. The down regulation of the gene can be measured by methods known in the art, including, quantitative PCR or RNA sequencing.

[272] In some embodiments, the gene editing system is used in combination with an exogenous nucleic acid to increase expression of a polynucleotide, for example, a polynucleotide comprising a sequence that has at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identity to one of SEQ ID NOS: 1-16, 67-70, or 90-96. In some embodiments, the endonuclease is a Cas endonuclease as described in TABLE 6A or TABLE 6B. In some embodiments, the Cas endonuclease is a Cas9 endonuclease. In some embodiments, the Cas9 endonuclease comprises a sequence that is at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent, at least 99 percent, or 100 percent identical to SEQ ID NO: 203. In some embodiments, the endonuclease comprises a nuclear localization signal. For example, in some embodiments, the endonuclease comprises a nuclear localization signal comprising a sequence that is at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identity to SEQ ID NO: 201.

[273] In some embodiments, the Cas endonuclease is encoded by a polynucleotide that is optimized for expression in a fungal cell from the Psilocybe genus. Codon optimization is a process used to improve gene expression and increase the translational efficiency of a gene of interest by accommodating codon bias of the fungal cell. In some embodiments, the polynucleotide comprises codons that frequently occur in the Psilocybe genus. In some embodiments, the endonuclease comprises a nuclear localization signal. In some embodiments, the Cas endonuclease is encoded by a polynucleotide that is at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identity to SEQ ID NO: 203.

[274] In some embodiments, an organism described herein can be genetically modified using an endonuclease. The endonuclease can be used to introduces a genetic modification into a genome of, for example, a fungal cell resulting in an increased amount of one or more desired alkaloids, and/or derivatives or analogs thereof, as compared to an amount of the same compound in a comparable control without a genetic modification. In some embodiments, the endonuclease can be a Cas endonuclease, e.g., a Cas 9 endonuclease. The endonuclease can be guided by a nucleic acid, such as, a guide RNA.

[275] Accordingly, in some embodiments described herein a genetic modification can involve introducing an exogenous nucleic acid into an organism and/or performing a gene deletion/disruption in the organism. In some instances, this can be accomplished using homologous recombination (HR), wherein selective markers conferring resistance to, for example, an antifungal compound, e.g., hygromycin or neomycin, can be used to replace or integrate within the target locus. While some fungi, e.g., Saccharomyces cerevisiae, may have a relatively high HR efficiency, gene disruption can be difficult for many other fungal organisms due to a low HR efficiency. Provided here are efficient, rapid, powerful, and economical gene manipulation tools such as CRISPR technology, which as described in certain embodiments herein, is optimized for use on fungal organisms. This technology can be used to enhance the efficiency of gene manipulation and integration of, for example, one or more exogenous nucleic acids into a fungal cell.

[276] In some embodiments, the exogenous nucleic acid results in the increased expression of a polynucleotide comprising a sequence that has at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identity to one of SEQ ID NOS: 19-30. In some embodiments, the endonuclease is a Cas endonuclease. In some embodiments, the Cas endonuclease is a Cas9 endonuclease. In some embodiments, the Cas9 endonuclease comprises a sequence that is at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent, at least 99 percent, or 100 percent identical to SEQ ID NO: 202. In some embodiments, the endonuclease comprises a nuclear localization signal. For example, in some embodiments, the endonuclease comprises a nuclear localization signal comprising a sequence that is at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identity to SEQ ID NO: 201.

[277] In some embodiments, the Cas endonuclease is encoded by a polynucleotide that is optimized for expression in a fungal cell from the Psilocybe genus. Codon optimization is a process used to improve gene expression and increase the translational efficiency of a gene of interest by accommodating codon bias of the fungal cell. In some embodiments, the polynucleotide comprises codons that frequently occur in the Psilocybe genus. In some embodiments, the endonuclease comprises a nuclear localization signal. In some embodiments, the Cas endonuclease is encoded by a polynucleotide that is at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identity to SEQ ID NO: 203. [278] In some embodiments, the expression of the gene editing system inside the fungal cell results in a genetic modification that leads to an increased, or decreased expression of the psychotropic alkaloid as compared to a comparable fungal cell without the gene editing system. In some embodiments, the psychotropic alkaloid one of N, N-dimethyltryptamine, N-acetyl- hydroxytryptamine, 4-hydroxy-L-tryptophan, 5 -hydroxy -L-tryptophan, 7-hydroxy-L-tryptophan, 4-phosporyloxy-N, N-dimethyltryptamine , serotonin or a derivative thereof, aeruginascin , 2-(4- Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium , 4-phosphoryloxy-N,N- dimethytryptamine, ketamine, melatonin or a derivative thereof, normelatonin, 3,4- Methylenedioxymethamphetamine, isatin, harmine, P-carboline, N, N-dimethyltryptamine (DMT) or a derivative thereof.

[279] In another aspect, this disclosure provides a gene editing system for modifying a fungal cell, wherein the gene editing system comprising an endonuclease and at least one guide polynucleotide, or one or more nucleic acids encoding said endonuclease and the at least one guide polynucleotide, wherein the guide polynucleotide comprises a sequence that can bind to a gene comprising a sequence that has at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent or 100 percent identity to one of SEQ ID NOS: 1-18, or 89-99. In some embodiments, the guide polynucleotide binds to a gene that comprises a sequence comprising one of SEQ ID NOS: 60-116. In some embodiments, the at least one guide polynucleotide binds to the gene within 100 bases of the sequence comprising one of SEQ ID NOS: 60-116. In some embodiments, the at least one guide polynucleotide binds to the gene at least partially overlapping the sequence comprising one of SEQ ID NOS: 60-116. In some embodiments, the at least one guide polynucleotide comprises a targeting sequence that is complementary to one of SEQ ID NOS: 60-116. In some embodiments, the at least one guide polynucleotide comprises a targeting sequence that binds to one of SEQ ID NOS: 60-116. In some embodiments, the composition further comprises a fungal cell from division Basidiomycota. In some embodiments, the fungal cell is a fungal protoplast.

[280] In some embodiments, the gene editing system comprises the one or more nucleic acids encoding the endonuclease and the at least one guide polynucleotide. In some embodiments, the endonuclease and the at least one guide polynucleotide are encoded on a single nucleic acid. In some embodiments, the endonuclease and the at least one guide polynucleotide are encoded on different nucleic acids. In some embodiments, the one or more nucleic acids comprises a gene promoter that drives expression of the endonuclease or the at least one guide polynucleotide in the fungal cell. In some embodiments, the gene promoter has a sequence that is at least 95 percent identical to any one of SEQ ID NOS: 88-90, or 93. In some embodiments, the one or more nucleic acids comprise a first gene promoter that drives expression of the endonuclease and a second gene promoter that drives expression of the at least one guide polynucleotide, wherein the first gene protomer is different from the second gene promoter. In some embodiments, the first gene promoter comprises a GDP gene promoter and the second gene promoter comprises a U6 gene promoter.

[281] In some embodiments, the endonuclease of the gene editing system is in a complex with the at least one guide polynucleotide. In some embodiments, the at least one guide polynucleotide comprises a sequence that can bind to a target sequence that has at least 95 percent or 99 percent identity to one of SEQ ID NOS: 60-116. In some embodiments, the gene editing comprises at least two guide polynucleotides. In some embodiments, the gene editing system further comprises an exogenous nucleic acid. In some embodiments, the exogenous nucleic acid comprises a psilocybin synthase gene. In some embodiments, the exogenous nucleic acid comprises any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif. In some embodiments, the endonuclease is a Cas endonuclease. In some embodiments, the Cas endonuclease is a Cas9 endonuclease. In some embodiments, the endonuclease is encoded by a polynucleotide optimized for expression in the Psilocybe genus by comprising codons that frequently occur in the Psilocybe genus. In some embodiments, the endonuclease comprises a nuclear localization signal. In some embodiments, the nuclear localization signal is optimized for expression in a fungal cell from the genus Psilocybe. In some embodiments, the at least one guide polynucleotide comprises an RNA.

[282] In another aspect, this disclosure provides a gene editing system for genetically modifying a fungal cell, wherein the gene editing system comprises at least one nucleic acid sequence encoding a guide polynucleotide that binds to a nucleic acid involved in expression of a psychotropic alkaloid, wherein the at least one nucleic acid sequence is operably linked to a first gene promoter and a nucleic acid sequence encoding an endonuclease operably linked to a second gene promoter, wherein the second gene promoter is distinct from the first gene promoter. The first gene promoter and the second gene promoter can have different promoter activities. The promoter activities of the different gene promoters can ensure that the endonuclease and the guide RNA are expressed at desired ratios inside the fungal cell. For example, in some embodiments, it may be desirable to express higher quantities of guide polynucleotide than endonuclease to ensure each endonuclease associates with a guide polynucleotide and thereby minimize off target activities of the endonucleases inside the fungal cell.

[283] In some embodiments, the first gene promoter is a fungal gene promoter. In some embodiments, the fungal gene promoter comprises a U6 gene promoter. In some embodiments, the U6 gene promoter comprises a sequence that has at least 95 percent identity to one of SEQ ID NOS: 400-403. In some embodiments, the second gene promoter a GPD gene promoter from Agaricus bisporus. In some embodiments, the GDP promoter comprises SEQ ID NO: 406.

[284] In some embodiments, the guide polynucleotide comprises a sequence that can bind to a target sequence that has at least 95 percent identify to any one of SEQ ID NOS: 60-116. In some embodiments, the composition further comprises a fungal cell. In some embodiments, the fungal cell is from division Basidiomycota. In some embodiments, the fungal cell is a fungal protoplast.

[285] In some embodiments, the composition further comprises an exogenous nucleic acid. In some embodiments, the exogenous nucleic acid comprises a psilocybin synthase gene. In some embodiments, the exogenous nucleic acid comprises any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin- related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helixloop-helix transcription factor that binds to an E-box motif.

[286] In some embodiments, the endonuclease provided with the composition is a Cas endonuclease. In some embodiments, the Cas endonuclease is a Cas9 endonuclease. In some embodiments, the endonuclease is encoded by a polynucleotide optimized for expression in the Psilocybe genus by comprising codons that frequently occur in the Psilocybe genus. In some embodiments, the endonuclease comprises a nuclear localization signal.

[287] In another aspect, this disclosure provides a composition, the composition comprising a vector encoding a gene editing system for genetically modifying a fungal cell, wherein the gene editing system comprises at least two guide polynucleotides that each bind to a nucleic acid involved in production or regulation of a psychotropic alkaloid, and an endonuclease. In some embodiments, wherein the vector comprises a binary agrobacterium vector. In some embodiments, the vector further comprises a first border sequence and a second border sequence flanking the sequence encoding the gene editing system, wherein the first border sequence and the second border sequence comprise a repeat that is associated with agrobacterium-mediated transformation. In some embodiments, the gene editing system comprises at least four guide polynucleotides. In some embodiments, the at least two polynucleotide, or the at least four guide polynucleotides, are positioned on the vector downstream of a fungal gene promoter. [288] In some embodiments, the fungal gene promoter is a U6 promoter comprising a sequence that has at least 95 percent identity to one of SEQ ID NOS: 400-403. In some embodiments, the endonuclease is regulated by a second gene promoter that is different from the fungal gene promoter. In some embodiments, the second gene promoter comprises a GPD gene promoter from Agaricus bisporus. In some embodiments, the endonuclease comprises a Cas9 endonuclease linked to a nuclear localization signal. In some embodiments, the polynucleotide sequence encoding the endonuclease is optimized comprises amino acid codons that frequently occur in the Psilocybe genus. In some embodiments, the nucleic acid involved in production or regulation of a psychotropic alkaloid comprises a sequence that encodes any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif.

[289] In some embodiments, the fungal gene promoter is a U6 promoter comprising a sequence that has at least about 95 percent identity to one of SEQ ID NOS: 400-403. In some embodiments, the endonuclease is regulated by a second gene promoter that is different from the fungal gene promoter. In some embodiments, the second gene promoter comprises a GPD gene promoter from Agaricus bisporus. In some embodiments, the endonuclease comprises a Cas9 endonuclease linked to a nuclear localization signal. In some embodiments, the polynucleotide sequence encoding the endonuclease is optimized comprises amino acid codons that frequently occur in the Psilocybe genus. In some embodiments, the nucleic acid involved in production or regulation of a psychotropic alkaloid comprises a sequence that encodes any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif.

[290] In some embodiments, the nucleic acid involved in production or regulation of a psychotropic alkaloid comprises a sequence that encodes any one of PsiD, PsiH, PsiH2, PsiK, PsiM, PsiTl, PsiT2, PsiR, and PsiP (PsiP as used herein, unless stated otherwise, refers to a PsiP phosphatase family gene or its protein expression product). When a fungus includes multiple PsiP genes, the genes or their protein expression products referenced herein may be numbered to differentiate, e.g., PsiPl and PsiP2. In some embodiments, one of the at least two guide polynucleotides comprise a sequence can bind to one of SEQ ID NOS: 60-116.

[291] In some embodiments, when the gene editing system is expressed in a fungal cell, the gene editing system results in increased expression of any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, a psilocybin-related phosphotransferase, or helix-loop-helix transcription factor that binds to an E- box motif, as compared to a comparable fungal cell without the gene editing system. In some embodiments, one of the at least two guide polynucleotides comprises a sequence that binds to a promoter or an enhancer of a psilocybin biosynthesis gene. In some embodiments, the psilocybin biosynthesis gene is selected from the group consisting of: PsiD, PsiK, PsiH, PsiM, PsiR, PsiP2, PsiPl, TrpE, TrpL and TrpM. In some embodiments, the composition further comprises an exogenous nucleic acid. In some embodiments, the exogenous nucleic acid comprises a psilocybin synthase gene. In some embodiments, the exogenous nucleic acid comprises any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif. In some embodiments, the exogenous nucleic acid comprises a sequence that has at least a 95 percent identity to any one of SEQ ID NOS: 1-18. In some embodiments, the exogenous nucleic acid results in the increased expression of a polynucleotide comprising a sequence that has at least 95 percent identity to one of SEQ ID NOS: 19-30.

[292] In another aspect, this disclosure provides a kit comprising a gene editing system as disclosed herein. The kit can further comprise instructions and one or more ancillary reagents.

[293] In another aspect, this disclosure provides a method comprising introducing a gene editing system as disclosed herein into a fungal cell. The gene editing system can comprise a polynucleotide guide RNA and an endonuclease, or one or more nucleic acids encoding the guide polynucleotide and the endonuclease. In some embodiments, the method further comprises expressing the gene editing system inside the fungal cell. In some embodiments, the fungal cell is from division Basidiomycota. In some embodiments, the gene editing system comprises the endonuclease and the guide polynucleotide, wherein the endonuclease is pre-complexed with the guide polynucleotide. In some embodiments, the gene editing system comprises the endonuclease and the guide polynucleotide, wherein the gene editing system is delivered as a vector into the fungal cell. In some embodiments, the gene editing system is introduced into the fungal cell with a bacterium. In some embodiments, the bacterium is Agrobacterium tumefaciens. In some embodiments, expression of the gene editing system in the fungal cell results in any one of increased, or decreased tryptophan decarboxylation, increased, or decreased tryptamine 4-hydroxylation, increased, or decreased 4-hydroxytryptamine O-phosphorylation, or increased, or decreased psilocybin production via sequential N-methylations in the fungal cell as compared to a comparable fungal cell without the gene editing system. In some embodiments, the expression of the gene editing system results in increased production of an alkaloid by the fungal cell as compared to a comparable fungal cell without the gene editing system, wherein the alkaloid comprises a compound selected from N,N-dimethyltryptamine, N-acetyl- hydroxytryptamine, 4-hydroxy-L-tryptophan, 5 -hydroxy -L-tryptophan, 7-hydroxy-L-tryptophan, 4-phosporyloxy-N,N-dimethyltryptamine , serotonin or a derivative thereof, aeruginascin , 2-(4- Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium , 4-phosphoryloxy-N,N- dimethytryptamine, ketamine, melatonin or a derivative thereof, normelatonin, 3,4- Methylenedioxymethamphetamine, isatin, harmine, P-carboline, N,N-dimethyltryptamine (DMT) or a derivative thereof.

[294] In another aspect, this disclosure provides a composition for genetically modifying a fungal organism, the composition comprising a vector comprising a nucleic acid sequence positioned between first and second border sequences, wherein the first and second border sequences are each cleavable by a VirDl or a VirD2 endonuclease, wherein the nucleic acid sequence encodes a gene editing system comprising at least one polynucleotide sequence encoding a guide RNA that binds to a nucleic acid involved in expression of an alkaloid biosynthesis gene product, wherein the at least one polynucleotide sequence is operably linked to a first gene promoter, and a polynucleotide sequence encoding a Cas endonuclease operably linked to a second gene promoter, wherein the second gene promoter is distinct from the first gene promoter. In some embodiments, the first gene promoter is a fungal gene promoter.

In some embodiments, the fungal gene promoter comprises an U6 promoter comprising a sequence that is at least 95 percent identical to any one of SEQ ID NOS: 400-403.

[295] In some embodiments, the second gene promoter comprises a gene promoter from a fungal organism. In some embodiments, the second gene promoter comprises a GPD gene promoter from Agaricus bisporus. In some embodiments, the vector comprises a binary agrobacterium vector. In some embodiments, the first and second border sequences comprise a 25-base pair repeat that is associated with agrobacterium-mediated transformation.

[296] In another aspect, this disclosure provides a method for genetically modifying a fungal cell, the method comprising preparing a CRISPR vector for delivery into a fungal cell, wherein preparing the CRISPR vector comprises combining at least two polynucleotides encoding a guide RNA with a polynucleotide encoding a Cas endonuclease into a bacterial vector, thereby forming the CRISPR vector integrating the CRISPR vector into a bacterium, and

[297] delivering the CRISPR vector to the fungal cell using the bacterium. In some embodiments, after preparing the CRISPR vector, the at least two polynucleotides encoding guide RNA and the polynucleotide encoding the Cas endonuclease are between a first and a second border sequence of the CRISPR vector. In some embodiments, the first and second border sequences comprise a 25-base pair repeat that are associated with agrobacterium- mediated transformation. In some embodiments, the at least two polynucleotides encoding the guide RNA comprise a fungal gene promoter upstream of a sequence that encodes the guide RNA. In some embodiments, the fungal gene promoter comprises a sequence that is at least about 95 percent identical to any one of SEQ ID NOS: 400-403, or 406. In some embodiments, the polynucleotide encoding the Cas endonuclease comprises a GDP gene promoter from Agaricus bisporus. In some embodiments, the bacterium is from Agrobacterium tumefaciens. In some embodiments, the fungal cell is from division Basidiomycota. In some embodiments, the fungal cell comprises a mycelium. In some embodiments, delivering the CRISPR vector to the fungal cell using the bacterium comprises culturing the fungal cell with the bacterium for 72-92 hours. In some embodiments, the guide RNA comprises a sequence that is complementary to a nucleic acid involved in expression of a gene product of an alkaloid biosynthesis pathway. In some embodiments, one of the at least two polynucleotides encoding the guide RNA comprises a sequence that can bind to one of SEQ ID NOS: 60-116. In some embodiments, the polynucleotide encoding the Cas endonuclease is optimized to include amino acid codons that frequently occur in Psilocybe cubensis. In some embodiments, the CRISPR vector is delivered with an exogenous nucleic acid that comprises a polynucleotide of interest desired to be incorporated into the fungal cell. In some embodiments, the polynucleotide of interest encodes a transgene selected from the group consisting of PsiD, PsiK, PsiH, PsiH2, PsiM, PsiR, PsiP2, PsiPl, TrpE, and TrpM. In some embodiments, the CRISPR vector is delivered with an exogenous nucleic acid that comprises a polynucleotide of interest desired to be incorporated into the fungal cell. In some embodiments, the polynucleotide of interest encodes a transgene selected from the group consisting of PsiD, PsiK, PsiH, PsiH2, PsiL, PsiM, PsiR, PsiP2, PsiPl, TrpE, and TrpM. In some embodiments, the transgene comprises a sequence that is at least 95 percent identical to any one of SEQ ID NOS: 1-18 or 89-99. In some embodiments, the bacterial vector is a binary agrobacterium vector. In some embodiments, the method further comprises expressing the at least two polynucleotides encoding guide RNA and the polynucleotide encoding the Cas endonuclease in the fungal cell, thereby producing a genetically modified fungal cell. In some embodiments, the genetically modified fungal cell comprises a higher concentration of any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin- related methyltransferase, psilocybin-related kinase, a psilocybin-related phosphotransferase, or a helix-loop-helix transcription factor that binds to an E-box motif, than a comparable fungal cell without the genetic modification. In some embodiments, the genetically modified fungal cell comprises a detectable increase in the expression of an alkaloid as compared to a comparable fungal cell without the genetic modification within 7-10 days of delivering the CRISPR vector to the fungal cell. In some embodiments, the alkaloid comprises any one of N-acetyl- hydroxytryptamine, 4-hydroxy-L-tryptophan, 5 -hydroxy -L-tryptophan, 7-hydroxy-L-tryptophan, 4-phosporyloxy-N,N-dimethyltryptamine , serotonin or a derivative thereof, aeruginascin , 2-(4- Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium , 4-phosphoryloxy-N,N- dimethytryptamine, ketamine, melatonin or a derivative thereof, normelatonin, 3,4- Methylenedioxymethamphetamine, isatin, harmine, P-carboline, N,N-dimethyltryptamine (DMT) or a derivative thereof. In some embodiments, preparing the CRISPR vector comprises binding a free end of one of the at least two polynucleotides with a free end of the bacterial vector, thereby linking the one of the at least two polynucleotides with the bacterial vector. In some embodiments, the free end of the at least two polynucleotides comprises an overhang sequence that is complementary to an overhang sequence of the bacterial vector. In some embodiments, the method comprises binding a free end of a second one of the at least two polynucleotides with a second free end of the one of the at least two polynucleotides thereby linking the second one of the at least two polynucleotides with the bacterial vector.

[298] In some embodiments, the free end of the second one of the at least two polynucleotides comprises an overhang sequence that is different than the overhang sequence of the free end of the one of the at least two polynucleotides.

[299] In some embodiments, the method further comprises binding a free end of the polynucleotide encoding the Cas endonuclease with a second free end of one of the at least two polynucleotides to thereby link the polynucleotide encoding the Cas endonuclease to the bacterial vector. In some embodiments, the free end of the polynucleotide encodes the Cas endonuclease comprises an overhang sequence that is different than the overhang sequence of the one of the at least two polynucleotides encoding the guide RNA.

[300] In one aspect, this disclosure provides methods and compositions for making genetically modified organisms with enhanced alkaloid production. The alkaloids can be new to nature alkaloids. The alkaloids can be new to the organism from which the alkaloids are produced. The alkaloids can be alkaloids that are rare.

[301] In some embodiments, this disclosure involves introducing a heterologous nucleic acid into a fungal organism, wherein the expression of the heterologous nucleic acid leads to the production of a new to nature alkaloid. In some embodiments, nucleic acid encodes a TrpM gene from the fungus Psilocybe serbica. In some embodiments, the TrpM gene is introduced into Psilocybe cubensis on an exogenous nucleic acid. The gene can comprise a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 94. Expression of the gene can be driven by a GDP promoter, e.g., SEQ ID NO: 406. By expressing the TrpM from the exogenous nucleic acid inside Psilocybe cubensis one or more alkaloids can be produced. In some embodiments, the one or more alkaloids are new to the organism from which they are produced. In some embodiments, the one or more alkaloids produced includes DMT.

[302] In some embodiments, this disclosure involves introducing a heterologous nucleic acid into a fungal organism, wherein the expression of the heterologous nucleic acid leads to the production of a new to nature alkaloid. In some embodiments, the nucleic acid encodes a PsiM gene from P. Azurescence, which has been described as being more efficient in producing psilocybin. Thus, in some embodiments wherein the gene is introduced into Psilocybe cubensis, the genetic modification can result in increased production of psilocybin. The gene can comprise a sequence that is at least about: 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 91. Expression of the gene can be driven by a GDP promoter, e.g., SEQ ID NO: 406.

[303] In some embodiments, as discussed below, a nucleic acid encoding an indolethylamine N- methyltransferase (INMT) gene from homo sapiens HsINMT can be incorporate into an organism. The gene can comprise a sequence that is at least about: 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 99. In some embodiments, a nucleic acid encoding an aromatic L- amino acid decarboxylase (AAAD) from P. cubensis can be incorporated into an organism. The gene can comprise a sequence that is at least about: 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 92. In some embodiments, a gene encoding strictosidine synthase (STST) from Catharanthus roseus, can be incorporated into an organism. The gene can comprise a sequence that is at least about: 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 95. In some embodiments, a McbB gene comprising a sequence that is at least about: 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 96 can be incorporated into an organism.

[304] In some embodiments, the nucleic acid involved in production or regulation of a psychotropic alkaloid comprises a sequence that encodes any one of PsiD, PsiH, PsiH2, PsiK, PsiM, PsiTl, PsiT2, PsiR, and PsiP (PsiP as used herein, unless stated otherwise, refers to a PsiP phosphatase family gene or its protein expression product). In some embodiments, the nucleic acid involved in production or regulation of a psychotropic alkaloid comprises a sequence that encodes any one of PsiD, PsiH, PsiH2, PsiK, PsiL, PsiM, PsiTl, PsiT2, PsiR, and PsiP (PsiP as used herein, unless stated otherwise, refers to a PsiP phosphatase family gene or its protein expression product). When a fungus includes multiple PsiP genes, the genes or their protein expression products referenced herein may be numbered to differentiate, e.g., PsiPl and PsiP2. In some embodiments, one of the at least two guide polynucleotides comprise a sequence can bind to one of SEQ ID NOS: 60-116.

[305] In some embodiments, when the gene editing system is expressed in a fungal cell, the gene editing system results in increased expression of any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, a psilocybin-related phosphotransferase, or helix-loop-helix transcription factor that binds to an E- box motif, as compared to a comparable fungal cell without the gene editing system. In some embodiments, one of the at least two guide polynucleotides comprises a sequence that binds to a promoter or an enhancer of a psilocybin biosynthesis gene. In some embodiments, the psilocybin biosynthesis gene is selected from the group consisting of: PsiD, PsiK, PsiH, PsiM, PsiR, PsiP2, PsiPl, TrpE, and TrpM. In some embodiments, the composition further comprises an exogenous nucleic acid. In some embodiments, the exogenous nucleic acid comprises a psilocybin synthase gene. In some embodiments, the exogenous nucleic acid comprises any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif. In some embodiments, the exogenous nucleic acid comprises a sequence that has at least a 95 percent identity to any one of SEQ ID NOS: 1-18. In some embodiments, the exogenous nucleic acid results in the increased expression of a polynucleotide comprising a sequence that has at least 95 percent identity to one of SEQ ID NOS: 19-30.

[306] In another aspect, this disclosure provides a kit comprising a gene editing system as disclosed herein. The kit can further comprise instructions and one or more ancillary reagents.

[307] In another aspect, this disclosure provides a method comprising introducing a gene editing system as disclosed herein into a fungal cell. The gene editing system can comprise a polynucleotide guide RNA and an endonuclease, or one or more nucleic acids encoding the guide polynucleotide and the endonuclease. In some embodiments, the method further comprises expressing the gene editing system inside the fungal cell. In some embodiments, the fungal cell is from division Basidiomycota. In some embodiments, the gene editing system comprises the endonuclease and the guide polynucleotide, wherein the endonuclease is pre-complexed with the guide polynucleotide. In some embodiments, the gene editing system comprises the endonuclease and the guide polynucleotide, wherein the gene editing system is delivered as a vector into the fungal cell. In some embodiments, the gene editing system is introduced into the fungal cell with a bacterium. In some embodiments, the bacterium is Agrobacterium tumefaciens. In some embodiments, expression of the gene editing system in the fungal cell results in any one of increased tryptophan decarboxylation, increased tryptamine 4- hydroxylation, increased 4-hydroxytryptamine O-phosphorylation, or increased psilocybin production via sequential N-methylations in the fungal cell as compared to a comparable fungal cell without the gene editing system. In some embodiments, the expression of the gene editing system results in increased production of an alkaloid by the fungal cell as compared to a comparable fungal cell without the gene editing system, wherein the alkaloid comprises a compound selected from N-acetyl-hydroxytryptamine, 4-hydroxy-L-tryptophan, 5-hydroxy-L- tryptophan, 7-hydroxy-L-tryptophan, 4-phosporyloxy-N,N-dimethyltryptamine , serotonin or a derivative thereof, aeruginascin , 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l- aminium , 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin or a derivative thereof, normelatonin, 3,4-Methylenedioxymethamphetamine, isatin, harmine, P-carboline, N,N- dimethyltryptamine (DMT) or a derivative thereof.

[308] In another aspect, this disclosure provides a composition for genetically modifying a fungal organism, the composition comprising a vector comprising a nucleic acid sequence positioned between first and second border sequences, wherein the first and second border sequences are each cleavable by a VirDl or a VirD2 endonuclease, wherein the nucleic acid sequence encodes a gene editing system comprising at least one polynucleotide sequence encoding a guide RNA that binds to a nucleic acid involved in expression of an alkaloid biosynthesis gene product, wherein the at least one polynucleotide sequence is operably linked to a first gene promoter, and a polynucleotide sequence encoding a Cas endonuclease operably linked to a second gene promoter, wherein the second gene promoter is distinct from the first gene promoter. In some embodiments, the first gene promoter is a fungal gene promoter.

[309] In some embodiments, the second gene promoter comprises a gene promoter from a fungal organism. In some embodiments, the second gene promoter comprises a GPD gene promoter from Agaricus bisporus. In some embodiments, the vector comprises a binary agrobacterium vector. In some embodiments, the first and second border sequences comprise a 25-base pair repeat that is associated with agrobacterium-mediated transformation.

[310] In another aspect, this disclosure provides a method for genetically modifying a fungal cell, the method comprising preparing a CRISPR vector for delivery into a fungal cell, wherein preparing the CRISPR vector comprises combining at least two polynucleotides encoding a guide RNA with a polynucleotide encoding a Cas endonuclease into a bacterial vector, thereby forming the CRISPR vector integrating the CRISPR vector into a bacterium, and [311] delivering the CRISPR vector to the fungal cell using the bacterium. In some embodiments, after preparing the CRISPR vector, the at least two polynucleotides encoding guide RNA and the polynucleotide encoding the Cas endonuclease are between a first and a second border sequence of the CRISPR vector. In some embodiments, the first and second border sequences comprise a 25-base pair repeat that are associated with agrobacterium- mediated transformation. In some embodiments, the at least two polynucleotides encoding the guide RNA comprise a fungal gene promoter upstream of a sequence that encodes the guide RNA. In some embodiments, the fungal gene promoter comprises a sequence that is at least 95 percent identical to any one of SEQ ID NOS: 400-403, or 406. In some embodiments, the polynucleotide encoding the Cas endonuclease comprises a GDP gene promoter from Agaricus bisporus. In some embodiments, the bacterium is from Agrobacterium tumefaciens. In some embodiments, the fungal cell is from division Basidiomycota. In some embodiments, the fungal cell comprises a mycelium. In some embodiments, delivering the CRISPR vector to the fungal cell using the bacterium comprises culturing the fungal cell with the bacterium for 72-92 hours. In some embodiments, the guide RNA comprises a sequence that is complementary to a nucleic acid involved in expression of a gene product of an alkaloid biosynthesis pathway. In some embodiments, one of the at least two polynucleotides encoding the guide RNA comprises a sequence that can bind to one of SEQ ID NOS: 60-116. In some embodiments, the polynucleotide encoding the Cas endonuclease is optimized to include amino acid codons that frequently occur in Psilocybe cubensis. In some embodiments, the CRISPR vector is delivered with an exogenous nucleic acid that comprises a polynucleotide of interest desired to be incorporated into the fungal cell. In some embodiments, the polynucleotide of interest encodes a transgene selected from the group consisting of PsiD, PsiK, PsiH, PsiH2. PsiM, PsiR, PsiP2, PsiPl, TrpE, and TrpM. In some embodiments, the transgene comprises a sequence that is at least 95 percent identical to any one of SEQ ID NOS: 1-18 or 89-99. In some embodiments, the bacterial vector is a binary agrobacterium vector. In some embodiments, the method further comprises expressing the at least two polynucleotides encoding guide RNA and the polynucleotide encoding the Cas endonuclease in the fungal cell, thereby producing a genetically modified fungal cell. In some embodiments, the genetically modified fungal cell comprises a higher concentration of any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, psilocybin-related kinase, a psilocybin-related phosphotransferase, or a helix-loop-helix transcription factor that binds to an E-box motif, than a comparable fungal cell without the genetic modification. In some embodiments, the genetically modified fungal cell comprises a detectable increase in the expression of an alkaloid as compared to a comparable fungal cell without the genetic modification within 7-10 days of delivering the CRISPR vector to the fungal cell. In some embodiments, the alkaloid comprises any one of N-acetyl-hydroxytryptamine, 4-hydroxy-L-tryptophan, 5 -hydroxy -L-tryptophan, 7- hydroxy-L-tryptophan, 4-phosporyloxy-N,N-dimethyltryptamine , serotonin or a derivative thereof, aeruginascin , 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium , 4- phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin or a derivative thereof, normelatonin, 3, 4-Methylenedi oxymethamphetamine, isatin, harmine, P-carboline, N,N- dimethyltryptamine (DMT) or a derivative thereof. In some embodiments, preparing the CRISPR vector comprises binding a free end of one of the at least two polynucleotides with a free end of the bacterial vector, thereby linking the one of the at least two polynucleotides with the bacterial vector. In some embodiments, the free end of the at least two polynucleotides comprises an overhang sequence that is complementary to an overhang sequence of the bacterial vector. In some embodiments, the method comprises binding a free end of a second one of the at least two polynucleotides with a second free end of the one of the at least two polynucleotides thereby linking the second one of the at least two polynucleotides with the bacterial vector.

[312] In some embodiments, the free end of the second one of the at least two polynucleotides comprises an overhang sequence that is different than the overhang sequence of the free end of the one of the at least two polynucleotides.

[313] In some embodiments, the method further comprises binding a free end of the polynucleotide encoding the Cas endonuclease with a second free end of one of the at least two polynucleotides to thereby link the polynucleotide encoding the Cas endonuclease to the bacterial vector. In some embodiments, the free end of the polynucleotide encodes the Cas endonuclease comprises an overhang sequence that is different than the overhang sequence of the one of the at least two polynucleotides encoding the guide RNA.

[314] In one aspect, this disclosure provides methods and compositions for making genetically modified organisms with enhanced alkaloid production. The alkaloids can be new to nature alkaloids. The alkaloids can be new to the organism from which the alkaloids are produced. The alkaloids can be alkaloids that are rare.

[315] In some embodiments, this disclosure involves introducing a heterologous nucleic acid into a fungal organism, wherein the expression of the heterologous nucleic acid leads to the production of a new to nature alkaloid. In some embodiments, nucleic acid encodes a TrpM gene from the fungus Psilocybe serbica. In some embodiments, the TrpM gene is introduced into Psilocybe cubensis on an exogenous nucleic acid. The gene can comprise a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 94. Expression of the gene can be driven by a GDP promoter, e.g., SEQ ID NO: 406. By expressing the TrpM from the exogenous nucleic acid inside Psilocybe cubensis one or more alkaloids can be produced. In some embodiments, the one or more alkaloids are new to the organism from which they are produced. In some embodiments, the one or more alkaloids produced includes DMT.

[316] In some embodiments, this disclosure involves introducing a heterologous nucleic acid into a fungal organism, wherein the expression of the heterologous nucleic acid leads to the production of a new to nature alkaloid. In some embodiments, the nucleic acid encodes a PsiM gene from P. Azurescence, which has been described as being more efficient in producing psilocybin. Thus, in some embodiments wherein the gene is introduced into Psilocybe cubensis, the genetic modification can result in increased production of psilocybin. The gene can comprise a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 91. Expression of the gene can be driven by a GDP promoter, e.g., SEQ ID NO: 406.

[317] In some embodiments, as discussed below, a nucleic acid encoding an indolethylamine N- methyltransferase gene from homo sapiens HsINMT can be incorporate into an organism. The gene can comprise a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 99. In some embodiments, a nucleic acid encoding an aromatic L-amino acid decarboxylase (AAAD) from P. cubensis can be incorporated into an organism. The gene can comprise a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 92. In some embodiments, a gene encoding strictosidine synthase (STST) from Catharanthus roseus, can be incorporated into an organism. The gene can comprise a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 95. In some embodiments, a McbB gene comprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 96 can be incorporated into an organism.

[318] CRISPR (clustered regularly interspaced short palindromic repeats) can refer to a family of DNA repeats found in certain bacterial genomes. In some instances, the CRISPR protein can include a Cas9 endonuclease, which can form a complex with a crRNA/tracrRNA hybrid to recognize, bind, and ultimately cleave foreign DNA at a target site. The crRNA/tracrRNA hybrid can be designed as a single guide RNA. For example, any one of the guide RNAs shown in Tables 9-17, which are identified with corresponding PAM sequences. For example, Table 9 discloses guide RNA targets with corresponding PAM sequences useful for making genetic modifications to PsiD. Table 10 discloses guide RNA targets with corresponding PAM sequences useful for making genetic modifications to PsiPl. Table 11 discloses guide RNA targets with corresponding PAM sequences useful for making genetic modifications to PsiP2. Table 12 discloses guide RNA targets with corresponding PAM sequences useful for making genetic modifications to TrpE. Table 13 discloses guide RNA targets with corresponding PAM sequences useful for making genetic modifications to TrpM. Table 14 discloses guide RNA targets with corresponding PAM sequences useful for making genetic modifications to PsiH. Table 15 discloses guide RNA targets with corresponding PAM sequences useful for making genetic modifications to PsiR. Table 16 discloses guide RNA targets with corresponding PAM sequences useful for making genetic modifications to intergenic sequences, e.g., fungal genomic DNA. Table 17 discloses guide RNA targets with corresponding PAM sequences useful for making genetic modifications to psilocybin synthase genes.

[319] In some embodiments, an organism described herein can be genetically modified using an endonuclease. The endonuclease can be used to introduces a genetic modification into a genome of, for example, a fungal cell resulting in an increased amount of one or more desired alkaloids, and/or derivatives or analogs thereof, as compared to an amount of the same compound in a comparable control without a genetic modification. In some embodiments, the endonuclease can be a Cas endonuclease, e.g., a Cas 9 endonuclease. The endonuclease can be guided by a nucleic acid, such as, a guide RNA. The guide RNA can be any one of the guide RNAs disclosed in TABLE 9, TABLE 10, TABLE 11, TABLE 12, TABLE 13, TABLE 14, TABLE 15, TABLE 16, or TABLE 17. The guide RNA can be complementary sequence to any one of the guide RNAs disclosed in TABLE 9, TABLE 10, TABLE 11, TABLE 12, TABLE 13, TABLE 14, TABLE 15, TABLE 16, or TABLE 17. The guide RNA can comprise a sequence that binds to a sequence disclosed in TABLE 9, TABLE 10, TABLE 11, TABLE 12, TABLE 13, TABLE 14, TABLE 15, TABLE 16, or TABLE 17. The guide RNA can comprise a sequence that binds to a sequence disclosed in TABLE 9, TABLE 10, TABLE 11, TABLE 12, TABLE 13, TABLE 14, TABLE 15, TABLE 16, or TABLE 17 under stringent conditions. The guide RNA can comprise a target sequence as disclosed in TABLE 9, TABLE 10, TABLE 11, TABLE 12, TABLE 13, TABLE 14, TABLE 15, TABLE 16, or TABLE 17. The target sequence can be at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to one of sequences disclosed in TABLE 9, TABLE 10, TABLE 11, TABLE 12, TABLE 13, TABLE 14, TABLE 15, TABLE 16, or TABLE 17

[320] In some embodiments, this disclosure provides a genetic modification can involve introducing an exogenous nucleic acid into an organism and/or performing a gene deletion/disruption in the organism. In some instances, this can be accomplished using homologous recombination (HR), wherein selective markers conferring resistance to, for example, an antifungal compound, e.g., hygromycin or neomycin, can be used to replace or integrate within the target locus. While some fungi, e.g., Saccharomyces cerevisiae, may have a relatively high HR efficiency, gene disruption can be difficult for many other fungal organisms due to a low HR efficiency. Provided here are efficient, rapid, powerful, and economical gene manipulation tools such as CRISPR technology, which as described in certain embodiments herein, is optimized for use on fungal organisms. This technology can be used to enhance the efficiency of gene manipulation and integration of, for example, one or more exogenous nucleic acids into a fungal cell.

[321] In some embodiments, an endonuclease system that is used to genetically modified an organism described herein comprises a CRISPR enzyme and a guide nucleic that hybridizes with a target sequence in, or adjacent to the gene or the promoter or enhancer associated therewith. In some cases, a target sequence can be at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides in length.

[322] In some embodiments, a target sequence can be at most 7 nucleotides in length. In some cases, a target sequence can hybridize with at least one of SEQ ID NOS: 1-16, a gene or a regulator element of a gene selected from Table 2. In some cases, a guide nucleic acid can be chemically modified. In an embodiment, a guide polynucleotide is a single guide RNA (sgRNA). In an embodiment, a guide nucleic acid can be a chimeric single guide comprising RNA and DNA. In some cases, a CRISPR enzyme can comprise or be a Cas protein or variant or derivative thereof.

Table 9. gRNA target sequences for PsiD

Table 10. gRNA target sequences for PsiPl

Table 11. gRNA target sequences for PsiP2.

Table 12. gRNA target sequences for TrpE.

Table 13. gRNA target sequences for TrpM.

TABLE 14. Exemplary gRNA targets + PAM sequences for PsiH

Table 15. gRNA target sequences +PAM sequences for PsiR.

Table 16. gRNA target sequences +PAM sequences for intergenic/watermark sequences.

Table 17. gRNA target sequences for gene editing of Psi genes.

[323] The recognition of a target DNA target region can depend on a protospacer adjacent motif (PAM) which can be located at the 3 '-terminus of a 20 bp target sequence. Once the CRISPR complex (e.g., Cas9 and associated guide RNA) recognizes the target DNA sequence, the CRISPR complex can generate a double strand break (DSB) at the DNA target locus. In some instances, one of two cellular DNA repair mechanisms, non-homologous end joining (NHEJ) and homologous recombination (HR), can play a role in precise genome editing and gene manipulation. For example, NHEJ, which is sometimes regarded as an error-prone repair mechanism that generates either short insertions or deletions of nucleotides in close proximity to the DSB site(s), can be used. If these short insertions or deletions exist in a gene coding region, or within a portion of the promoter involved in recruiting proteins involved in transcription, the function of the endogenous gene, for example a gene encoding psilocybin phosphatase, can be disrupted. Consequently, this procedure can be used for generating gene mutations. In other embodiments, a homology independent targeted integration (HITI) strategy can be used which allows fragments (e.g., exogenous nucleic acids) to be integrated into the genome by NHEJ repair. [324] Various versions of CRISPR systems can be used. In some instances, the CRISPR system can be introduced into the genome of a target organism using Agrobacterium tumefaciens- mediated transformation. When the expression of Cas protein and guide RNA can be under the control of either a constitutive or inducible promoter. For example, in some embodiments, the Cas protein is under the control of a GDP gene protomer, while the guide RNA is under the control of a U6 gene promoter. In some instances, the Cas protein is optimized for use in a fungal cell. Exemplary promoter sequences are shown in TABLE 18. More specifically, Table 18 lists promoter sequences useful for driving transgene expression in fungi. The promoter sequences include a GPD promoter from Agaricus pisporus including the ATG (bolded), the first intron and the first six base pairs. The gene promoters further include U6 promoters from P. Cubensis.

Table 18. Exemplary Promoters

[325] In some embodiments, an endonuclease system that is used to genetically modified an organism described herein comprises a CRISPR enzyme and a guide nucleic that hybridizes with a target sequence in, or adjacent to the gene or the promoter or enhancer associated therewith. In some cases, a target sequence can be at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides in length.

[326] In some embodiments, a target sequence can be at most 7 nucleotides in length. In some cases, a target sequence can hybridize with at least a portion of a sequence that is complementary to one of SEQ ID NOS: 1-16 or 120-128. In some cases, a guide nucleic acid can be chemically modified. In an aspect, a guide polynucleotide is a single guide RNA (sgRNA). In an aspect, a guide nucleic acid can be a chimeric single guide comprising RNA and DNA. In some cases, a CRISPR enzyme can comprise or be a Cas protein or variant or derivative thereof. In some cases, a Cas protein comprises Cast, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, CasSt, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, CseSe, Csci, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csrn4, Csm5, Csm6, Cmr, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csf 1, Csf2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, C2cl, C2c2, C2c3, Cpfl, CARF, DinG, homologues thereof, or modified versions thereof. In some cases, a Cas protein can be a Cas9. In some cases, Cas9 is a modified Cas9 that binds to a canonical PAM. In some cases, Cas9 recognizes a non-canonical PAM. In some cases, a guide polynucleotide binds a target sequence 3-10 nucleotides from a PAM. In some cases, a CRISPR enzyme coupled with a guide polynucleotide can be delivered into a genetically modified organism as an RNP. In some cases, a CRISPR enzyme coupled with a guide polynucleotide can be delivered into a genetically modified organism by a mRNA encoding the CRISPR enzyme and the guide polynucleotide.

[327] In some embodiments, the endonuclease comprises at least one nuclear localization signal. In some embodiments, the endonuclease is delivered into a cell as a functional protein, e.g., a ribonucleoprotein, wherein the protein comprises a nuclear localization size and associated with a guide RNA. In other instances, an exogenous nucleic acid encoding at least one RNA-guided endonuclease comprising at least one nuclear localization signal is introduced into the cell of the organism for genetic modification.

[328] Accordingly, in some embodiments, a CRISPR enzyme coupled with a guide polynucleotide can be delivered into a genetically modified organism by a vector comprising a nucleic acid encoding the CRISPR enzyme and the guide polynucleotide. In an embodiment, a vector further comprises a selection marker or a reporter gene. In some embodiments, a RNP, complex, or vector can be delivered via agrobacterium mediated transformation, biolistic particle bombardment, or protoplast transformation. In some embodiments, a RNP, mRNA, or vector further comprises a donor polynucleotide or a nucleic acid encoding the donor polynucleotide. In an embodiment, a donor polynucleotide (e.g., an exogenous nucleic acid) comprises homology to sequences flanking a target sequence. In an aspect, a donor polynucleotide further comprises a barcode, a reporter gene, or a selection marker.

[329] In some embodiments, the exogenous nucleic acid is incorporated in a plasmid. In some cases, the plasmid ispGWB5 or pGHGWY. In some embodiments, the plasmid is delivered into said genetically modified organism via electroporation, microinjection, mechanical cell deformation, lipid nanoparticles, AAV, lentivirus, agrobacterium mediated transformation, biolistic particle bombardment, or protoplast transformation. In some cases, the plasmid further comprises a barcode, a reporter gene, or a selection marker. In some cases, the plasmid further comprises a promoter. In some cases, the promoter is 35S, GPD, EFla, Actin, or CcDEDl. The promoter may be a promoter selected from TABLE 18.

[330] In some embodiments, a genetic modification can be conducted by contacting a cell of an organism with a gene editing system. In some embodiments, the gene editing system comprises a Cas endonuclease enzyme, a TALE-nuclease, transposon-based nuclease, a zinc finger nuclease, meganuclease, argonaute, Mega-TAL or DNA guided nuclease. In some embodiments, the geneediting system comprises a DNA-guided nuclease with an argonaute.

[331] In some embodiments, a gene-editing system that is used to make a genetic modification to a fungal cell comprises an active nucleic acid guided endonuclease. In some embodiments, the active nucleic acid guided endonuclease comprises a Cas endonuclease, such as a Cas9 endonuclease, complexed with a guide RNA. In some embodiments, the guide RNA comprises a target sequence selected from Tables 9-table 17. In some embodiments, the guide RNA binds to a gene comprising a target sequence shown in Tables 9-table 17, and SEQ ID NOS: 60-116. In some embodiments, the guide RNA within about 100 bases, about 75 bases, about 50 bases, about 25 bases, about 5 bases, or 1 base of the sequence comprising one of SEQ ID NOS: 60- 116. In some embodiments, the guide RNA binds to the gene at a loci at least partially overlapping the sequence comprising one of SEQ ID NOS: 60-116. In some embodiments, the at least one guide polynucleotide comprises a targeting sequence that is complementary to one of SEQ ID NOS: 60-116. In some embodiments, the at least one guide polynucleotide comprises a targeting sequence that binds to one of SEQ ID NOS: 60-116.

[332] In some embodiments, the Cas endonuclease complex is delivered into the fungal cell in the form of an active ribonuclease protein. In some embodiments, the Cas endonuclease complex is integrated into the fungal cell by incubating the fungal cell with the Cas endonuclease complex in combination with a reagent. In some embodiments, the reagent comprises a detergent. In some embodiments, the reagent comprises a nonionic surfactant. In some embodiments, the nonionic surfactant comprises Triton X-100. In some embodiments, the fungal cell is a fungal protoplast. In some embodiments, the fungal protoplast is incubated with the Cas endonuclease complex for at least 1 hour at 20 degrees Celsius. In some embodiments, the fungal cell is screened for the genetic modification 72 hours post genetic modification.

[333] In some embodiments, an endonuclease is delivered with a guide polynucleotide to target the endonuclease to an endogenous nucleic acid of the organism. In some embodiments, the guide polynucleotide binds to the endogenous nucleic acid and ultimately cleaves the endogenous nucleic acid at the target site. Cleavage of the endogenous nucleic acid at the target site can result in a deleterious deletion of a protein encoded by the nucleic acid. In some embodiments, a guide polynucleotide is used to target a gene, e.g., a psilocybin synthase gene for knockdown or knockout. The gene can be a psilocybin synthase gene. The gene can be one of the genes listed in Tables 1 and 2. The gene can be one of the genes listed in TABLE 1, TABLE 2, TABLE SATABLE 3B The gene can be targeted by designing a guide polynucleotide that is complementary to a portion of one of the genes listed in Tables 1 and 2. The guide polynucleotide can target, for example, a gene comprising a sequence of one of SEQ ID NOS: 60-116. Accordingly, in some embodiments an endonuclease is used to knockdown or knock out one of PsiD, PsiK, PsiM, PsiPl, PsiP2, PsiH, PsiH2, PsiR, TrpD, TrpE, or any combination thereof. PsiP as used herein, unless stated otherwise, refers to a PsiP phosphatase family gene or its protein expression product. When a fungus includes multiple PsiP genes, the genes or their protein expression products referenced herein may be numbered to differentiate, e.g., PsiPl and PsiP2.

[334] In some embodiments, the modification can reduce expression of psilocybin phosphatase by at least 50% as compared to a comparable fungal cell without said modification. For example, the modification can reduce the expression of psilocybin phosphatase by 50%, 60%, 70%, 80%, 90%, 95%, or 100% as compared to a comparable fungal cell without said modification. By reducing the expression of psilocybin phosphatase, the modification can result in a decreased expression of psilocin in the engineered fungal cell as compared to a comparable fungal cell without the modification, e.g., a fungal cell from Psilocybe cubensis with wild-type normal expression level of psilocybin phosphatase. Accordingly, the modification can result in an increased expression of psilocybin in the engineered fungal cell as compared to a comparable fungal cell without the modification.

[335] In some embodiments, the engineered fungal cell further comprises a second modification that results in at least one of: increased tryptophan decarboxylation, increased tryptamine 4-hydroxylation, increased 4-hydroxytryptamine O-phosphorylation, or increased psilocybin production via sequential N-methylations as compared to a comparable fungal cell without the second modification. For example, the engineered fungal cell can further comprise a second modification that results in an increased expression of a gene product as compared to a comparable fungal cell without the second modification, wherein the gene product is encoded by a gene selected from the group consisting of PsiD, PsiM, PsiH, PsiH2, PsiK, and PsiR. For example, the engineered fungal cell can further comprise a second modification that results in an increased expression of a gene product as compared to a comparable fungal cell without the second modification, wherein the gene product is encoded by a gene selected from the group consisting of PsiD, PsiM, PsiH, PsiH2, PsiL, TrpE, TrpM, PsiK, and PsiR. The gene product can include, for example, at least a portion of any amino acid listed in TABLE 4A-TABLE 4B.

[336] In some embodiments, the second modification can be an exogenous nucleic acid that is incorporated into the engineered fungal cell, wherein the exogenous nucleic acid includes a sequence that is at least 80%, 90%, 95%, or 100% identical to any one of SEQ ID NOS: 1-19 or 90-98. In other instances, the exogenous nucleic acid includes a sequence that is at least 75%, 80%, 85%, 90%, 99% or 100% identical to any one of SEQ ID NOS: 1-19 or 90-98. In some instances, the exogenous nucleic acid includes a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to one of the sequences of TABLE 2, TABLE 3 A, or TABLE 3B.

[337] In some embodiments, any one of SEQ ID NOS: 1-28 comprises a base edit. In some embodiments, the any one of SEQ ID NOS: 1-28 is incorporated using a Cas protein or Cas fusion protein.

[338] As a result of the genetic modification, the engineered fungal cell may express a gene product by at least 6-fold greater than as expressed in a comparable fungal cell without the second modification. As a result of the genetic modification, the engineered fungal cell may express a gene product by at least 6-fold lesser than as expressed in a comparable fungal cell without the second modification. For example, the engineered fungal cell may express a gene product (e.g., mRNA encoding tryptophan decarboxylase) by at least 10-fold greater than as expressed in a comparable wild-type fungal cell. To assess the expression of the gene product, a qPCR or western blot analysis can be performed.

[339] The exogenous nucleic acid can include a gene promoter that is positioned upstream of the gene for which upregulated expression is desired. The gene can be any one of PsiD, PsiM, PsiH, PsiK, or PsiR. The gene can be any one of PsiD, PsiM, PsiH, PsiK, PsiL, TrpE, TrpM, or PsiR The gene promoter can be any one of a 35S promoter, a GDP promoter, or a CcDEDl promoter.

[340] In some embodiments, this disclosure provides a pharmaceutical composition comprising the engineered fungal cell or an extract thereof. The pharmaceutical composition can include an effective amount of the engineered fungal cell or the extract thereof for treating a health condition. The composition can be formulated such that an effective amount of the composition for treatment of the health condition can be delivered in a single dose format. In other instances, this disclosure provides a supplement comprising an extract of the engineered fungal cell. In yet other instances, this disclosure provides a food supplement comprising an extract of the engineered fungal cell.

[341] The modification can be accomplished by contacting a fungal cell, e.g., a fungal protoplast, with a gene editing system. The gene editing system can be any one of a Cas endonuclease, an agrobacterium-mediated insertion of exogenous nucleic acid, TALE-nuclease, a transposon-based nuclease, a zinc finger nuclease, a mega nuclease, a mega-TAL or DNA guided nuclease. For example, in some instances the gene editing system is a nucleic acid guided endonuclease (e.g., Cas9), which is delivered into the fungal cell as in the format of an active ribonucleoprotein. The gene editing system can be delivered into the fungal cell in an active form with, for example, a detergent such as Triton X-100. In some embodiments, the geneediting system includes a nuclear localization signal to facilitate the passage of the gene-editing system into a nucleus of the fungal cell.

In an aspect, this disclosure provides gene editing systems, compositions, and methods for making a genetic modification to enhance the production of alkaloids. In some embodiments, the genetic modification is introduced by a gene-editing system, such as the CRISPR system. In some embodiments, the genetic modification involves an exogenous nucleic acid that is integrated into the genome of a target organism. The exogenous nucleic acid can encode a psilocybin synthase gene. The exogenous nucleic acid can encode a gene identified in TABLE 1, TABLE 2, or TABLE 3A-TABLE 3B. In some embodiments, the genetic modification results in the production of a homologous polypeptide. In some embodiments, the genetic modification results in the increased expression of a polynucleotide that is involved in the regulation or expression of an alkaloid. For example, in some embodiments, the genetic modification results in the expression of a polypeptide selected from TABLE 1, TABLE 2, or TABLE 3A-TABLE 3B. In some embodiments, the genetic modification makes use of a gene editing system to add or remove one or more nucleotides, wherein the addition or removal of the one or more nucleotides results in a deleterious mutation that reduces or elements expression of the targeted nucleic acid. In some embodiments, the gene editing system is directed to a target nucleic acid using a guide polynucleotide, e.g., one or more of the guide polynucleotides that are complementary to, or capable of hybridizing with, a gene comprising a sequence selected from TABLE 9-TABLE 17. In some embodiments, the target nucleic acid is a psilocybin synthase gene. For example, in some embodiments, the target nucleic acid is a gene identified in TABLE 1, TABLE 2, TABLE 3A-TABLE 3B. In some embodiments, the target nucleic acid is a regulatory element of a gene, such as, an enhancer or a promoter.

[342] In particular, the genetically modified organism hosting the illustrated pathway can be described as having a genetic modification that results in an increased expression of L-try ptophan decarboxylase 123 as compared to a comparable wild-type organism. The genetic modification can be achieved by using a gene editing system to integrate an exogenous nucleic acid into the genome of a fungal cell. The exogenous nucleic acid can encode one or more copies of PsiD. For example, the exogenous nucleic acid can be integrated into the genome of the fungal cell by using the gene editing system, e.g., a Cas endonuclease and a guide polynucleotide, to cleave fungal DNA at a target sequence and incorporate the exogenous nucleic acid using the fungal cell’s endogenous repair pathways.

[343] When expressed in the genetically modified organism, the L-try ptophan decarboxylase 123 can convert L-tryptophan 106 and/or 4-hydroxy-L-tryptophan 108 into tryptamine 107 or 4- hydroxy tryptamine 109, respectively. Advantageously, the upregulated expression of L- tryptophan decarboxylase 123 can result in the increased production of 4-hydroxytryptamine 109 and/or tryptamine 107. Because, as illustrated, 4-hydroxytryptamine 109 and tryptamine 107 can be precursors to compounds including psilocybin 101, baecystin 103, norbaeocystin 104, aeruginascin 105, psilocin 102, and N,N-dimethyltryptamine 110, the increased expression of L- tryptophan decarboxylase 123 can result in the increased production of any one or more of psilocybin 101, baecystin 103, norbaeocystin 104, aeruginascin 105, psilocin 102, tryptamine 107, 4-hydroxytryptamine 109, or N,N-di methyltryptamine 1 10. The genetic modification can involve introducing one or more additional copies of a gene encoding L-tryptophan decarboxylase 123 into the genome of the organism. The one or more additional copies can be introduced using a CRISPR system. The CRISPR system can include a guide polynucleotide that directs the CRISPR system to a target nucleic acid. The guide polynucleotide can be, for example, one of the guide polynucleotides listed in TABLE 9- TABLE 17. The CRISPR system, when in contact with the target, nucleic acid, may make a double stranded break in the target nucleic acid. A donor sequence comprising the exogenous nucleic acid can be integrated at the break.

[344] FIG. 2 shows a biosynthesis pathway that is genetically modified to downregulate expression of psilocybin phosphatase 225. The genetic modification can result, in the increased production of an alkaloid, psilocybin. The exemplary' pathway can be hosted by a genetically modified organism that is, for example, a fungal cell from the genus Psilocybe. The genetically modified organism can be genetically modified to reduce or eliminate expression of psilocybin phosphatase 225, which when expressed in a wild-type fungal cell from the genus Psilocybe converts psilocybin 201 into psilocin 202.

[345] This disclosure provides a CRISPR toolkit that can be used to make genetic modifications to an alkaloid biosynthesis pathway so as to produce new' or rare alkaloids.

[346] FIG. 4 is a schematic representation of a CRISPR system used for genetic engineering. The illustrated system possess multiplex capabilities and can be used to clone up to 4 guide RNAs in a single destination vector for gene editing in Psilocybe cubensis. The gRNAs can be designed using Psilocybe cubensis sequence available on NCBI, for example, (https://www.ncbi.nlm.nih.gOv/assembly/GCA_017499595.l). Suitable guides can be ordered as oligo pairs with specific overhangs as described below.

[347] In particular, disclosed is a CRISPR vector toolkit for making one or more genetic modifications to an organism. In some embodiments, the organism is a fungal organism. The fungal organism can be a fungal protoplast. The CRISPR vector toolkit includes 6 entry vectors (pMGA, pMGB, pMGC, pMGD, pMGE, pMGF). The backbone of the vectors can be a commercially available vector, pUC57. Exemplary vector sequences are shown in Table 15 below with vector names shown in parenthesis.

[348] Making reference to FIG. 4., vectors pMGA, B, C and D can be used to clone gRNAs directly downstream of a Psilocybe cubensis U6 promoter. Exemplary U6 promoters are described below. Vector pMGE can consist of a strong promoter driving a Psilocybe cubensis codon optimized Cas9 gene or the nickase variant (D10 A, nCas9). Table 14 shows optimal codons for designing Cas endonucleases, and NLS sequences for use in Psilocybe cubensis. Such codon optimization can be used to improve gene expression and increase the translational efficiency of the Cas9 by accommodating codon bias of the fungal cell. The Psilocybe cubensis codon optimized Cas9 gene may comprise a sequence that is at least 75 percent, 80 percent, 85 percent, 90 percent, 95 percent, 99 percent, or 100 percent identical to SEQ ID NO.: 202, which can achieve high expression and translation efficiency in fungal cells.

[349] Vector pMGF can consist of a strong promoter driving a hygromycin resistance gene. The hygromycin resistance gene is useful during selection. Vectors PMGA,B,C,D,E and F can be assembled together into a binary destination vector (pMGZ) flanked by a left and right border suitable for agrobacterium-mediated Psilocybe cubensis transformation. An agrobacterium can be used to integrate the plasmid system into a fungal cell. In other instances, the Vectors PMGA,B,C,D,E and F can be integrated into a commercial plasmid (e.g., pUC57 or a viral plasmid) and delivered into a fungal cell without agrobacterium.

[350] Vectors, pMGA, B, C and D can be used to clone guide RNAs of interest. The guide RNAs can be inserted directly downstream of a Psilocybe cubensis U6 promoter and directly upstream of a guide RNA scaffold sequence, see below. The guide RNA of interest may comprise a sequence that binds with an alkaloid synthase gene. The following elements can be part of the pMGA, B, C and D vectors: Bsal site (forward orientation), Overhang 1 for assembly in destination vector, U6 promoter, Overhang A for gRNA insertion, BbsI site (reverse orientation), ccdb negative selection marker, BbsI site (forward orientation), Overhang B for gRNA insertion, Guide RNA scaffold sequence, U6 terminator, Overhang 2 for assembly in destination vector, and Bsal site (reverse orientation). The Bsal site can comprise sequence GGTCTC (forward, 5’ ->3’ orientation) and sequence GAGACC (reverse, 5 ’->3’ orientation). Exemplary pMGA, B, C, and D plasmid sequences are provided in Table 19.

[351] The pMGA, B, C and D vectors may be identical with the exception of the overhangs for assembly in destination vector. Overhang sequences used to produce the vectors is described in Table 12. Differential overhang sequences can be used to ensure inclusion of all guides into a final vector.

[352] Table 19 Vector overhang sequences.

[353] The U6 promoter can comprise any one of pU6-l, pU6-2 and pU6-l 1, described below. Guide entry vectors for all 3 promoters were designed and named pMGA-1, pMGB-1, pMGC-1, pMGD-1, pMGA-2, pMGB-2, pMGC-2, pMGD-2, pMGA-11, pMGB-11, pMGC-11 and pMGD-11.

[354] pU6-l promoter:

CGATTTCTTTAGGGCCGTAGGCTAGTAATCATCGACCGTTTTAATCATTAATGTACTT AGACAATAAATATAAGATGCAATACAAGTCAATGGGAGAAACTAGACTTTACAAAA CCTTTAAAAGCCCTGGTGAGATATGAGAAGGTTTATGACAGAATATATCGCCATTAA TGTGAGGTTGTGGACACTGCTGGTAGTCAAGGCTGCCCGTGAACCATATTTAGTCAC ATGTAATCACCCCGCGTGCTAAACAAAAAGCAAAATATCAGTAAGATAGTCACAGT CATAACACTGTTGAAT (SEQ ID NO: 400)

[355] >pU6-2 promoter:

TGCCAAAAAGCCTTCTTGTGGCCTGCTTACTATTAAGGCAACTAATTCAAGAACAAG TGATTCTGGGTAGGTAGATGCCACAGTTCATGATAATAAAGGCGAAGTCAGAAGGA GTAGTCCGTTGATGAAGAAAGCAGAAGGCAAGGAATGTTGGTGGCTTTTGGTTGCG GTAGCACTGAAACCGTGTCCGGACTTCGCCGGGAGCAGACAATGGCTTGGTTGGAT TACATAATAATACCCCGCGGGCCAGACAATATTCAAAATCCTAACAAAGATGTCTC AGGTAATACATTCGCTAAT (SEQ ID NO: 401)

[356] pU6-l l promoter:

GGTACCAGCAGTACCAGCACCAGCCACTGCATTATTGAATCTGACATCTGCAACAGC AAGGTACAATTTTTGTTTTACATTTTACTCATTAATATTAGCACCTATAGCTGTGGCC AATCTTTTGACGACGACTCTCTCACGCTGGAGGAAAGCATGGTACGGGCATTAATTG CCAGCGTAGAACAAGCGTAGGATATGGGCAACCTCGCTGATTTCTATATTTGGTAAG AAGTCTCACCCCGTGAGCTAAGCAAAAAGCAAAACCCTTGCTATGTCAACATCCCA CTGCCATACACTATT (SEQ ID NO: 403)

[357] FIG. 5 shows an alignment of three U6 promoters used for gene editing. U6 is highlighted between residues 282 and 420. A conserved region ca 40 nt upstream of the three U6 promoters is highlighted between residues 222 and 281.

[358] Overhangs for guide insertion are as follow (Table 20). Note that the last four nucleotide in the original sequence of the U6-1 promoter overhang is GTTT. Because it can be the same as the scaffold overhang, the sequence can be changed to GAAT to avoid recircularization of the plasmid without a guide, thereby enhancing cloning efficiency.

[359] Table 20. Overhangs sequences for guide insertion

[360] BbsI site can be GAAGAC (forward, 5’->3’ orientation) and GTCTTC (reverse, 5’->3’ orientation). Ccdb negative selection marker can allow selection for vectors with guide RNA inserted in place of ccdb by transforming a ccdb sensitive E. coli strain (eg DH5a). Sequence of ccdb (in bold), including promoter and terminator sequence, is as follows: gccggatccagtgctaacatggtctagaaggaggtcagctatgcagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtg gatgtacagagtgatattattgacacgcccgggcgacggatggtgatccccctggccagtgcacgtctgctgtcagataaagtctcccgtga actttacccagtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccagtctccgttatcggggaagaa gtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataactgcagaggaggtaatcaa (SEQ ID NO: 404)

[361] Sequence of guide RNA scaffold, including U6 terminator (TTTTTT) is as follows: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA AAGTGGCACCGAGTCGGTGCTTTTTT (SEQ ID NO: 405).

[362] The guides can be ordered as a pair of oligos with additional nucleotides at the 5’ end to generate overhangs allowing their oriented insertion in the entry vector directly downstream of the U6 promoter and directly upstream of the scaffold sequence (see overhangs A and B sequence in Table 13). Note that a “G” or a “C” can be added after the four nucleotides overhangs specific of the U6 promoters of the forward oligo or at the end of the reverse oligo, respectively, to increase transcription mediated by RNA Pol III.

[363] 6 shows an illustration on guide oligo design. The forward oligo starts with a 4 nucleotide specific of the U6 promoter used (highlighted NNNN) followed by a “G” (highlighted) and the 20 nucleotide sequence of the guide. The reverse oligo starts with the reverse of the 4 nucleotide sequence at the start of the scaffold (highlighted CAAA) followed by the reverse complement sequence of the guide ending with “C” (highlighted).

[364] The pMGE vector can comprise a Bsal site (forward orientation), Overhang 1 for assembly in destination vector, GPD (Glyceraldehyde-3 -phosphate dehydrogenase) promoter from Agaricus bisporus including the ATG, the first intron and the first six base pairs. Codon optimized 3x FLAG tag followed by a linker Codon optimized nuclear localization signal followed by a linker, a codon optimised Cas9 nuclease or Cas9 nickase (D10A), a Codon optimized linker followed by a nuclear localization signal, a 35S terminator sequence, an Overhang 2 for assembly in destination vector, and Bsal site (reverse orientation). The Bsal site is GGTCTC (forward, 5’->3’ orientation) and GAGACC (reverse, 5’->3’ orientation). The overhangs for assembly in destination vector pMGE are: Overhang 1 = CTGC, Overhang 2 = ACTA. Exemplary pMGE plasmid sequences are disclosed in Table 15 below.

[365] The GPD promoter can be selected from Agaricus bisporus and includes an ATG, the first intron and the first six base pairs is as follow (the ATG is indicated in bold).

[366] GAGCTCTGAAAGACGCAGCCGACGGTAAACACCCGGGCATCGAGAAAGGCA TTGTCGACTATACGGAAGAAGACGTTGTTTCCACCGATTTCGTTGGGAGCAACTATT CGATGATCTTTGACGCAAAAGCGGGCATCGCGTTGAACTCGCGTTTTATGAAATTAG TTGCATGGTATGATAATGAGTGGGGATATGCGCGTAGAGTCTGCGATGAGGTTGTGT ATGTAGCGAAGAAGAATTAAGAGGTCCGCAAGTAGATTGAAAGTTCAGTACGTTTT TAACAATAGAGCATTTTCGAGGCTTGCGTCATTCTGTGTCAGGCTAGCAGTTTATAA GCGTTGAGGATCTAGAGCTGCTGTTCCCGCGTCTCGAATGTTCTCGGTGTTTAGGGG TTAGCAATCTGATATGATAATAATTTGTGATGACATCGATAGTACAAAAACCCCAAT TCCGGTCACATCCACCATCTCCGTTTTCTCCCATCTACACACAACAAGCTCATCGCCg gtaccATGGTTTGTCTCTCGCTTGCATACCACCCAGCAGCTCACTGATGTCGACTTGTA GGTTAAA (SEQ ID NO: 406)

[367] Table 21 Shows the codon usage of Psilocybe cubensis, which was determined using CDS sequences retrieved on NCBI.

[368] The Sequence of the codon optimized 3x FLAG tag followed by a linker is as follows: gattataaagatcatgatggagattataaagatcatgatatcgattataaagatgatgatgataaagcagca (SEQ ID NO: 200)

[369] Sequence of the codon optimized nuclear localization signal followed by a linker is as follows: ccaaaaaaaaaaagaaaagtcggaatccatggagtcccagcagca (SEQ ID NO: 202)

[370] The sequence of the codon optimized Cas9 nuclease is as follows (note that the sequence of Cas9 nickase is identical except for the GAT codon highlighted in mutated to GCA): gataaaaaatattcaatcggattggatatcggaacaaactcagtcggatgggcagtcatcacagatgaatataaagtcccatcaaaaaaattc aaagtcttgggaaacacagatagacattcaatcaaaaaaaacttgatcggagcattgttgttcgattcaggagaaacagcagaagcaacaag attgaaaagaacagcaagaagaagatatacaagaagaaaaaacagaatctgctatttgcaagaaatcttctcaaacgaaatggcaaaagtc gatgattcattcttccatagattggaagaatcattcttggtcgaagaagataaaaaacatgaaagacatccaatcttcggaaacatcgtcgatg aagtcgcatatcatgaaaaatatccaacaatctatcatttgagaaaaaaattggtcgattcaacagataaagcagatttgagattgatctatttgg cattggcacatatgatcaaattcagaggacatttcttgatcgaaggagatttgaacccagataactcagatgtcgataaattgttcatccaattg gtccaaacatataaccaattgttcgaagaaaacccaatcaacgcatcaggagtcgatgcaaaagcaatcttgtcagcaagattgtcaaaatca agaagattggaaaacttgatcgcacaattgccaggagaaaaaaaaaacggattgttcggaaacttgatcgcattgtcattgggattgacacc aaacttcaaatcaaacttcgatttggcagaagatgcaaaattgcaattgtcaaaagatacatatgatgatgatttggataacttgttggcacaaat cggagatcaatatgcagatttgttcttggcagcaaaaaacttgtcagatgcaatcttgttgtcagatatcttgagagtcaacacagaaatcacaa aagcaccattgtcagcatcaatgatcaaaagatatgatgaacatcatcaagatttgacattgttgaaagcattggtcagacaacaattgccaga aaaatataaagaaatcttcttcgatcaatcaaaaaacggatatgcaggatatatcgatggaggagcatcacaagaagaattctataaattcatc aaaccaatcttggaaaaaatggatggaacagaagaattgttggtcaaattgaacagagaagatttgttgagaaaacaaagaacattcgataa cggatcaatcccacatcaaatccatttgggagaattgcatgcaatcttgagaagacaagaagatttctatccattcttgaaagataacagagaa aaaatcgaaaaaatcttgacattcagaatcccatattatgtcggaccattggcaagaggaaactcaagattcgcatggatgacaagaaaatca gaagaaacaatcacaccatggaacttcgaagaagtcgtcgataaaggagcatcagcacaatcattcatcgaaagaatgacaaacttcgata aaaacttgccaaacgaaaaagtcttgccaaaacattcattgttgtatgaatatttcacagtctataacgaattgacaaaagtcaaatatgtcaca gaaggaatgagaaaaccagcattcttgtcaggagaacaaaaaaaagcaatcgtcgatttgttgttcaaaacaaacagaaaagtcacagtca aacaattgaaagaagattatttcaaaaaaatcgaatgcttcgattcagtcgaaatctcaggagtcgaagatagattcaacgcatcattgggaac atatcatgatttgttgaaaatcatcaaagataaagatttcttggataacgaagaaaacgaagatatcttggaagatatcgtcttgacattgacatt gttcgaagatagagaaatgatcgaagaaagattgaaaacatatgcacatttgttcgatgataaagtcatgaaacaattgaaaagaagaagata tacaggatggggaagattgtcaagaaaattgatcaacggaatcagagataaacaatcaggaaaaacaatcttggatttcttgaaatcagatg gattcgcaaacagaaacttcatgcaattgatccatgatgattcattgacattcaaagaagatatccaaaaagcacaagtctcaggacaaggag attcattgcatgaacatatcgcaaacttggcaggatcaccagcaatcaaaaaaggaatcttgcaaacagtcaaagtcgtcgatgaattggtca aagtcatgggaagacataaaccagaaaacatcgtcatcgaaatggcaagagaaaaccaaacaacacaaaaaggacaaaaaaactcaag agaaagaatgaaaagaatcgaagaaggaatcaaagaattgggatcacaaatcttgaaagaacatccagtcgaaaacacacaattgcaaaa cgaaaaattgtatttgtattatttgcaaaacggaagagatatgtatgtcgatcaagaattggatatcaacagattgtcagattatgatgtcgatcat atcgtcccacaatcattcttgaaagatgattcaatcgataacaaagtcttgacaagatcagataaaaacagaggaaaatcagataacgtccca tcagaagaagtcgtcaaaaaaatgaaaaactattggagacaattgttgaacgcaaaattgatcacacaaagaaaattcgataacttgacaaaa gcagaaagaggaggattgtcagaattggataaagcaggattcatcaaaagacaattggtcgaaacaagacaaatcacaaaacatgtcgca caaatcttggattcaagaatgaacacaaaatatgatgaaaacgataaattgatcagagaagtcaaagtcatcacattgaaatcaaaattggttt cagatttcagaaaagatttccaattctataaagtcagagaaatcaacaactatcatcatgcacatgatgcatatttgaacgcagtcgtcggaac agcattgatcaaaaaatatccaaaattggaatcagaattcgtctatggagattataaagtctatgatgtcagaaaaatgatcgcaaaatcagaa caagaaatcggaaaagcaacagcaaaatatttcttctattcaaacatcatgaacttcttcaaaacagaaatcacattggcaaacggagaaatc agaaaaagaccattgatcgaaacaaacggagaaacaggagaaatcgtctgggataaaggaagagatttcgcaacagtcagaaaagtcttg tcaatgccacaagtcaacatcgtcaaaaaaacagaagtccaaacaggaggattctcaaaagaatcaatcttgccaaaaagaaactcagata aattgatcgcaagaaaaaaagattgggatccaaaaaaatatggaggattcgattcaccaacagtcgcatattcagtcttggtcgtcgcaaaag tcgaaaaaggaaaatcaaaaaaattgaaatcagtcaaagaattgttgggaatcacaatcatggaaagatcatcattcgaaaaaaacccaatc gatttcttggaagcaaaaggatataaagaagtcaaaaaagatttgatcatcaaattgccaaaatattcattgttcgaattggaaaacggaagaa aaagaatgttggcatcagcaggagaattgcaaaaaggaaacgaattggcattgccatcaaaatatgtcaacttcttgtatttggcatcacattat gaaaaattgaaaggatcaccagaagataacgaacaaaaacaattgttcgtcgaacaacataaacattatttggatgaaatcatcgaacaaatc tcagaattctcaaaaagagtcatcttggcagatgcaaacttggataaagtcttgtcagcatataacaaacatagagataaaccaatcagagaa caagcagaaaacatcatccatttgttcacattgacaaacttgggagcaccagcagcattcaaatatttcgatacaacaatcgatagaaaaaga tatacatcaacaaaagaagtcttggatgcaacattgatccatcaatcaatcacaggattgtatgaaacaagaatcgatttgtcacaattgggag gagat (SEQ ID NO: 202)

[371] The sequence of the codon optimized linker followed by a nuclear localization signal: ggaatccatggagtcccagcagcaccaaaaaaaaaaagaaaagtctga (SEQ ID NO: 203)

[372] The 35S terminator sequence is as follow:

AGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTG TGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGA GCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAA TTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATC (SEQ ID NO: 204

[373] The following elements can be part of the pMGF vectors: a Bsal site (forward orientation), Overhang 1 for assembly in destination vector, a 35S promoter. Hygromycin resistance gene, a 35S terminator sequence, an Overhang 2 for assembly in destination vector, a Bsal site (reverse orientation). The Bsal site is GGTCTC (forward, 5’->3’ orientation) and GAGACC (reverse, 5’->3’ orientation) Overhangs for assembly in destination vector (Table 22).

[374] Table 22. Exemplary overhangs for destination vector.

[375] 35S promoter sequence can comprise a sequence as follows:

TGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGC

TATCTGTCACTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCC

ATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCA

AAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACG

TCTTCAAAGCAAGTGGATTGATGTGATAACATGGTGGAGCACGACACTCTCGTCTAC

TCCAAGAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCTATTGAGACTTTTCA

ACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTT

CATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATA

AAGGAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCC

CCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCA

AGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCC

TTCGCAAGACCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGA AATCACCAGTCTCTCTCTACAAATCTATCTCTCTCGAGCTTTCGCAGATCCCGGGGG GCAATGAGAT (SEQ ID NO: 205).

[376] Hygromycin resistance can comprise sequence:

ATGAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTT

CGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAG

CTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTT

CTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGA

AGTGCTTGACATTGGGGAGTTTAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGC

ACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTACAACC

GGTCGCGGAGGCTATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGT

TCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATAT

GCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCA GTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCG AAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATG GCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATAC GAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACG CGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCACGACTCCGGGCGTA TATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGA TGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGA CTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGT GTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAA GAAATAG (SEQ ID NO: 206)

[377] 35 S terminator sequence can be as follow: AGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTG TGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGA GCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAA TTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATC (SEQ ID NO: 207)

[378] Vectors, pMGA, B, C and D can be combined and ligated with pMGE and pMGF into a destination vector. The destination vector can include left and right border sequences that can be recognized by an endonuclease, such as, a VirDl and/or VirD2 enzyme, inside an agrobacterium. Inside the agrobacterium, vectors pMGA, B, C and D, pMGE and pMGF, can be excised. The border sequence may comprise 25 bp border sequences that act as a nicking site for endonuclease. For example, VirDl, a site-specific helicase, unwinds double-stranded DNA. A nuclease, VirD2, cuts the bottom strand of DNA from the right and left border, becoming singlestranded linear DNA, which is exported out of the bacterium and into the fungal cell by enzymes.

[379] Inside the fungal cell, the gene editing system can introduce one or more genetic modifications. The gene editing system can comprise an endonuclease and at least one guide polynucleotide, or one or more nucleic acids encoding said endonuclease and the at least one guide polynucleotide, wherein the endonuclease forms a complex with the guide polynucleotide to binds to a nucleic acid present in the fungal cell and alter production on an alkaloid. The gene editing system can further include and a reagent that facilitates incorporation of the gene editing system into the fungal cell. In some embodiments, the fungal cell is a fungal protoplast. In some embodiments, the fungal cell is from the genus Psilocybe. Modulation of DMT in Genetically Modified Fungi

[380] N, N-dimethyltryptamine (DMT) as described herein can be involved in the psilocybin biosynthesis pathway. Additional approaches to the production of DMT by genetically modified fungi are available by exploiting well-described metabolic and proteomic gene pathways. As described herein, a number of downstream alkaloids are produced by genomic pathways responsible for the biosynthesis of tryptophan. DMT is one such downstream alkaloid of tryptophan. One genomic pathway of particular interest involves targeting indolethylamine-N- methyltransferase (INMT) and TrpM gene sequences. In some embodiments, an engineered fungus described herein comprises over expression of DMT. In some embodiments, an engineered fungus described herein comprises increased production of DMT and an overexpression of a PsiD gene. In some embodiments, an engineered fungus described herein comprises an over expression of an INMT gene and an overexpression of a PsiD gene. In some embodiments, an engineered fungus described herein comprises an over expression of an INMT gene and reduced expression of a PsiH gene. In some embodiments, an engineered fungus described herein comprises an over expression of an INMT gene and reduced expression of a PsiH2 gene. In some embodiments, an engineered fungus described herein comprises an over expression of an INMT gene and reduced expression of a PsiH and a PsiH2 gene. In some embodiments, an engineered fungus described herein produces an increased amount of a rare alkaloid described herein.

[381] In some embodiments, an engineered fungus described herein comprises an over expression of an HsINMT gene and an overexpression of a PsiD gene. In some embodiments, an engineered fungus described herein comprises an over expression of an HsINMT gene and reduced expression of a PsiH gene. In some embodiments, an engineered fungus described herein comprises an over expression of an HsINMT gene and reduced expression of a PsiH2 gene. In some embodiments, an engineered fungus described herein comprises an over expression of an HsINMT gene and reduced expression of a PsiH and a PsiH2 gene. In some embodiments, an engineered fungus described herein produces an increased amount of a rare alkaloid described herein. In some embodiments the HsINMT gene is optimized for psilocybe fungi. In some embodiments the HsINMT gene is comprised in an Ustilago mays optimized sequence.

[382] In some embodiments, an engineered fungus described herein comprises an over expression of an PcINMT gene and an overexpression of a PsiD gene. In some embodiments, an engineered fungus described herein comprises an over expression of an PcINMT gene and reduced expression of a PsiH gene. In some embodiments, an engineered fungus described herein comprises an over expression of an PcINMT gene and reduced expression of a PsiH2 gene. In some embodiments, an engineered fungus described herein comprises an over expression of an PcINMT gene and reduced expression of a PsiH and a PsiH2 gene. In some embodiments, an engineered fungus described herein produces an increased amount of a rare alkaloid described herein. In some embodiments the PcINMT gene is optimized for psilocybe fungi. In some embodiments the PcINMT gene is comprised in an Ustilago mays optimized sequence.

[383] In some embodiments, an engineered fungus described herein comprises an over expression of an ZflNMT gene and an overexpression of a PsiD gene. In some embodiments, an engineered fungus described herein comprises an over expression of an ZflNMT gene and reduced expression of a PsiH gene. In some embodiments, an engineered fungus described herein comprises an over expression of an ZflNMT gene and reduced expression of a PsiH2 gene. In some embodiments, an engineered fungus described herein comprises an over expression of an ZflNMT gene and reduced expression of a PsiH and a PsiH2 gene. In some embodiments, an engineered fungus described herein produces an increased amount of a rare alkaloid described herein. In some embodiments the ZflNMT gene is optimized for psilocybe fungi. In some embodiments the ZflNMT gene is comprised in an Ustilago mays optimized sequence.

[384] In some embodiments, an engineered fungus described herein comprises an over expression of a plant INMT gene and an overexpression of a PsiD gene. In some embodiments, an engineered fungus described herein comprises an over expression of a plant INMT gene and reduced expression of a PsiH gene. In some embodiments, an engineered fungus described herein comprises an over expression of a plant INMT gene and reduced expression of a PsiH2 gene. In some embodiments, an engineered fungus described herein comprises an over expression of a plant INMT gene and reduced expression of a PsiH and a PsiH2 gene. In some embodiments, an engineered fungus described herein produces an increased amount of a rare alkaloid described herein. In some embodiments a plant INMT gene is optimized for psilocybe fungi. In some embodiments the plant INMT gene is comprised in an Ustilago mays optimized sequence.

[385] In some embodiments, an engineered fungus described herein produces an increased amount of a novel alkaloid described herein. In some embodiments, an engineered fungus described herein produces an increased amount of DMT. In some embodiments, an engineered fungus described herein produces an increased amount of a harmala alkaloid. In some embodiments, an engineered fungus described herein produces an increased amount of a harmala alkaloid and an increased production of DMT. In some embodiments, a plasmid used herein comprises a human INMT gene. In some embodiments the optimized human INMT gene for psilocybe cubensis comprises:

ATGAAAGGAGGATTCACAGGAGGAGATGAATATCAAAAACATTTCCTCCCAAGAGA TTATCTCGCAACATATTATTCTTTCGATGGATCTCCATCTCCAGAAGCAGAAATGCTC AAATTCAACCTCGAATGCCTCCATAAAACATTCGGACCAGGAGGACTCCAAGGAGA TACACTCATCGATATCGGATCTGGACCAACAATCTATCAAGTCCTCGCAGCATTCGA TTCTTTCCAAGATATCACACTCTCTGATTTCACAGATAGAAACAGAGAAGAACTCGA AAAATGGCTCAAAAAAGAACCAGGAGCATATGATTGGACACCAGCAGTCAAATTCG CATGCGAACTCGAAGGAAACTCTGGAAGATGGGAAGAAAAAGAAGAAAAACTCAG AGCAGCAGTCAAAAGAGTCCTCAAATGCGATGTCCATCTCGGAAACCCACTCGCAC CAGCAGTCCTC

[386] CCACTCGCAGATTGCGTCCTCACACTCCTCGCAATGGAATGCGCATGCTGCTC TCTCGATGCATATAGAGCAGCACTCTGCAACCTCGCATCTCTCCTCAAACCAGGAGG ACATCTCGTCACAACAGTCACACTCAGACTCCCATCTTATATGGTCGGAAAAAGAGA ATTCTCTTGCGTCGCACTCGAAAAAGAAGAAGTCGAACAAGCAGTCCTCGATGCAG GATTCGATATCGAACAACTCCTCCATTCTCCACAATCTTATTCTGTCACAAACGCAG CAAACAACGGAGTCTGCTTCATCGTCGCAAGAAAAAAACCAGGACCA. In some embodiments, the optimized human INMT gene for psilocybe cubensis compri ses : atgaagggcggcttcaccggcggcgacgagtaccagaagcacttcctcccccgcgactacctcgccacctactactcgtt cgacggctcgccctcgcccgaggccgagatgctcaagttcaacctcgagtgcctccacaagaccttcggccccggcggcctccagggcg acaccctcatcgacatcggctcgggccccaccatctaccaggtcctcgccgccttcgactcgttccaggacatcaccctctcggacttcacc gaccgcaaccgcgaggagctcgagaagtggctcaagaaggagcccggcgcctacgactggacccccgccgtcaagttcgcctgcgag ctcgagggcaactcgggccgctgggaggagaaggaggagaagctccgcgccgccgtcaagcgcgtcctcaagtgcgacgtccacctc ggcaaccccctcgcccccgccgtcctccccctcgccgactgcgtcctcaccctcctcgccatggagtgcgcctgctgctcgctcgacgcct accgcgccgccctctgcaacctcgcctcgctcctcaagcccggcggccacctcgtcaccaccgtcaccctccgcctcccctcgtacatggt cggcaagcgcgagttctcgtgcgtcgccctcgagaaggaggaggtcgagcaggccgtcctcgacgccggcttcgacatcgagcagctc ctccactcgccccagtcgtactcggtcaccaacgccgccaacaacggcgtctgcttcatcgtcgcccgcaagaagcccggcccctag.

[387] In some embodiments, a plasmid used herein comprises a zebrafish INMT gene. In some embodiments the optimized zebrafish INMT gene for psilocybe cubensis comprises:

ATGAGTGAATGCACAAACTTCACAGAAGGAGAATTCTATCAGGCACATTTTGACCC GCGTGCTTATGTCAGGAATTTCTACTCCAGCCCTCGAGGACACTCCGACGAAAAGGA TTTCCTTACTTTTGTTTTAGGGGTCTTCAGTAGATTATTTTCAACTGGGAAACACAGA GGGCAAAGGTTGATAGACGTGGGGAGCGGACCATCAATCCACTGCGTCATTAGCGC CTGCGCACACTATGACGAGATTCTTCTGTCTGATTTCTCTGACAACAATCGTAGAGA AATTGAAAAATGGCTAAAAAACCAAGAAGGGTGTCTAGATTGGAGTCCCATCCTCC AGCACGTTAGTAAAACGGAGGGGAAAAGACCGTCCGATTTAGAGGCTACGCTGAAG CAAAGAATCAAAAAGGTTTTAAAATGTGACGTCCGCCTGGAGAATCCGTTTGATCC GCTGACACTGGAACCAGCTGACTGTGTCATTACATCTCTGTGCTTGGAAGCAGCCTG TAAAGACATGCAGATATACCGCCAGGCTTTACATGGGTTGACCAAGCTCCTGTGTCC CGGTGGACTATTCGTCATGGTGGGTGTTCTGAGTGAAACCTTCTACAAGGTGGATGA ACAGCTCTTTTCTTGTCTTAGCCTCAAACAGAATGATATCGAGGAAGCACTGAAAGG TTTTGGCTTCTCTATCCAAGAGTTTAATGTACTACCTGCTGAAGACCAAAACAATTCT GTGTCTGACTTTGAGGCCGTTTTTGTTCTTGTGGCGACCAAGAACATCTGA. In some embodiments, incorporation of an INMT gene described herein into a plasmid is operably linked to a promoter. In some embodiments, incorporation of an INMT gene described herein into a plasmid is operably linked to a terminator. In some embodiments, the terminator sequence comprises:

AGTAGATGCCGACCGGATCTGTCGATCGACAAGCTCGAGTTTCTCCATAATAATGTG TGAGTAGTTCCCAGATAAGGGAATTAGGGTTCCTATAGGGTTTCGCTCATGTGTTGA GCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAA TTTCTAATTCCTAAAACCAAAATCCAGTACTAAAATCCAGATC.

[388] Psilocybe serbica can mono- and dimethylate L-tryptophan. PsTrpM. In some embodiments, L tryptophan can be metabolically decarboxylated through an L-amino acid decarboxylase (AAAD) gene product. In some embodiments, N,N-dimethyltryptophan (DMTP) is produced by a genetically engineered fungus. In some embodiments, DMTP is metabolically converted to DMT, 5 -hydroxy -DMT, or bufotenine. In some embodiments, a genetically modified fungus described herein comprises multiple copies of a transgene described herein. In some embodiments, genetically engineered fungus described herein comprises multiple copies of a transgene described herein. In some embodiments, the transgene is TrpM. In some embodiments, TrpM expression in a genetically engineered fungus is compared the TrpM expression in a comparable wild type fungus. In some embodiments, TrpM expression is evaluated using a molecular ladder comparing a wild-type psilocybe fungus with a DMTP expression fungus (FIG. 31 A). In some embodiments, TrpM expression is evaluated in arbitrary units as shown in (FIG. 3 IB).

[389] In some embodiments, sequence constructs for gene synthesis and subsequence plasmid preparation can be prepared using gene synthesis methods. In some embodiments, a hygromycin resistance vector can be used as a selection marker in the plasmid. Exemplary vector constructs for alkaloid modulation are shown in TABLE 23A-TABLE 23D. Exemplary vectors used for DMT production in a genetically modified organism are shown in TABLE 24. In some embodiments, human INMT (HsINMT) is optimized for expression in fungi (e.g, Psilocybe and Utsilago codon-optimized versions). In some embodiments, PcINMT is optimized for expression in fungi. In some embodiments, RT-PCR amplification of the full coding sequence of PcINMT which included approximately 789 base pairs. In some embodiments, PcINMT expression is evaluated in arbitrary units as shown in FIGs. 31A-31B.

[390] In some embodiments, a construct is used. In some embodiments a fungal construct is used. In some embodiments, a construct or a zebrafish construct is used. In some embodiments, a plant construct or a Xenopus laevis (XI) construct is used. In some embodiments, a primate construct is used. In some embodiments, a human construct is used. In some embodiments, a construct which can be a testing constructs is used at about: -20 degrees Celsius, -10 degrees Celsius, 0 degrees Celsius, 10 degrees Celsius, 25 degrees Celsius, 30 degrees Celsius, 40 degrees Celsius, 50 degrees Celsius, 60 degrees Celsius, 70 degrees Celsius, or up to about 80 degrees Celsius. In some embodiments, amino acids from a PsiD gene can affect INMT protein production. In some embodiments, Psilocybe Kozak sequences result in transgene overexpression in Psilocybe. In some embodiments, Psilocybe Kozak sequences result in transgene under-expression in Psilocybe. In some embodiments, a Kozak sequence is a consensus sequence.

TABLE 23A. Exemplary testing constructs to product DMT in fungi

[391] In some embodiments, selected contiguous amino acids from a PsiD gene product are used to modulate INMT production levels in a genetically modified organism. In some embodiments the contiguous amino acids are a chain of: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous amino acids from a PsiD gene. In some embodiments, gene product levels are measured in a mycelium sample of genetically modified organism. In some embodiments INMT inhibition can result from DMT production. In some embodiments allosteric inhibitory binding sites of DMT are removed. In some embodiments, Asp28 (D) and/or Glu34(E) are mutated to and alanine residue. In some embodiments this results in an INMT protein sequence with D28A and/or E34A mutations.

TABLE 23B. Exemplary testing constructs to product DMT in fungi

[392] In some embodiments, Psilocybe serbica possessed gene products that can monomethylate and dimethylate L-tryptophan. There are other Psilocybe species that do not have gene products to monomethylate and dimethylate L-tryptophan. This originates from a retained ancient duplication event of a portion of the egtDB gene (latter required for ergothioneine biosynthesis). Phylogenetically, this is unrelated to PcPsiM production. In some embodiments, DMTP (N,N,dimethyl-L-tryptophan) can be decarboxylated metabolically into DMT after ingestion in human body through the action of aromatic L-amino acid decarboxylase (AAAD). Many fungal species fungi also have AAAD proteins. In some embodiments, DMTP can metabolize to DMT in P.cubensis fruiting body. In some embodiments, the above metabolic process can provide for indirect DMT production in fungi.

TABLE 23C. Exemplary testing constructs to product DMT in fungi

[393] Harmala alkaloids inhibit monoamine oxidase (MAO) which can degrade psilocybin and DMT in human body. In some embodiments, psilocybin and DMT are present in genetically modified fungi described herein. In some embodiments, are fungal species which produce harmala alkaloids (i.e., harmala alkaloids such as harmane and harmine) but at very low amounts (around 0.2 pg/g). In some embodiments, a genetically modified fungi can produce harmala alkaloids. In some embodiments, a genetically modified fungi can product a harmala alkaloid in a higher concentration than the amount produced by a naturally occurring fungus of the same species. In some embodiments, the genetically modified fungus is a Psilocybe fungus. In some embodiments, the genetically modified fungus is a Psilocybe fungus and can produce a harmala alkaloid in a higher concentration than the amount produced by a naturally occurring fungus of the same psilocybe species. In nature, harmala alkaloids are a component of the entourage of alkaloids in Psilocybe fungi. In some embodiments, a gene native, or not native, to a fungus can produce a P-carboline scaffold. In bacteria, P-carboline scaffold is produced from L-tryptophan. In plants, condensation of tryptamine and secologanin (monoterpene) to produce a tetrahydro-P- carboline scaffold.

TABLE 23D. Exemplary testing constructs to product DMT in fungi

TABLE 24. Exemplary vector for DMT and INMT modulation in Fungi

[394] Psilocybe serbica mono- and dimethylates L-tryptophan, are unlike many other Psilocybe fungi. PsTrpM originates from a retained ancient duplication event of a portion of the egtDB gene (latter required for ergothioneine biosynthesis) and is phylogenetically unrelated to PcPsiM.

[395] In some embodiments, DMTP (N,N,dimethyl-L-tryptophan) can be decarboxylated metabolically into DMT after ingestion in human body through the action of aromatic L-amino acid decarboxylase (AAAD). In some embodiments, but because fungi also have AAAD proteins, DMTP can be metabolized to DMT in a P.cubensis fruiting body. In some embodiments, DMT production occurs from an indirect biosynthesis pathway alternative to the Psilocybin biosynthesis pathway.

[396] In some embodiments, the genetically modified organism produces an elevated amount of N,N, -dimethyltryptamine in comparison to a naturally occurring otherwise equivalent genetically modified organism. In some embodiments the genetically modified organism expresses a gene product as shown in TABLE 25.

TABLE 25. Exemplary Gene Products for DMT modulation in Genetically Modified Fungi

[397] To increase production of a bioactive compound, e.g., psilocybin, the bioactive compound is produced simultaneously with an inhibitor of its own degradation, thereby increasing the overall production of the bioactive compound. An enzyme that produces a P- carboline core is overexpressed in Psilocybe cubensis to enhance production of bioactive compounds.

[398] P-carbolines are neuroactive compounds that inhibit monoamine oxidases which degrade psilocybin in human body. They are present in P. cubensis (i.e., harmala alkaloids such as harmane and harmine) but at very low amounts (around 0.2 pg/g). They are part of entourage in Psilocybe to prevent psilocybin degradation in human body.

[399] FIGS. 21A and 21B shows P-carbolines biosynthesis pathways. P-carboline core construction requires a Pictet-Spengler cyclization process. FIG. 21A shows a pathway from bacteria, which is used to produce a P-carboline scaffold from L-tryptophan. FIG. 21B shows a related pathway from a plant, which involves condensation of tryptamine and secologanin to produce a tetrahydro-P-carboline compound.

[400] The P-carbolines biosynthesis pathway diverged from the same building block (Trp) as psilocybin but produces dissimilar compounds; yet contribute to the same pharmacological effects. Harmala alkaloids are also found in the Banisteriopsis caapi vine, the key plant ingredient in the sacramental beverage Ayahuasca.

[401] An enzyme that produces a P-carboline core is overexpressed in Psilocybe cubensis to enhance production of bioactive compounds. The enzyme is encoded by a bacterial gene, McbB gene, which has SEQ ID NO: 126. To induce expression of the McbB gene ins a fungal cell, the gene is driven by a GDP promoter with SEQ ID NO: 31. The induced expression of the McbB gene the fungal cell leads to production of a useful alkaloid, DMT (N, N-Dimethyltryptamine), which when delivered to patients suffering from mental health disorder results in reduced symptoms.

[402] Additional transgenic fungi are genetically modified to induce expression of a plant enzyme that produces P-carboline in Psilocybe cubensis. This can result in the enhanced production of DMT, which is found in some plant species. The enzyme is encoded by the plant gene, strictosidine synthase (STST) from Catharanthus roseus, which has SEQ ID NO: 125. Induced expression of the STST gene leads to production of DMT in the fungal cell.

[403] In some embodiments, Psilocybe cubensis is genetically modified to produce DMT. DMT is found in several plants and is one of the active ingredients in Ayahuasca. DMTP (N,N,dimethyl-L-tryptophan) can be decarboxylated metabolically into DMT after ingestion through the action of aromatic L-amino acid decarboxylase (AAAD). In some embodiments, this disclosure includes the discovery that PsTrpM, and in particular, PsTrpM from Psilocybe serbica, This can be used to produce DMT in a genetically modified Psilocybe cubensis.

[404] FIG. 22 illustrates the methyl transfer steps of TrpM and PsiM during biosynthetic pathways to N,N-dimethyl-L-tryptophan and psilocybin, respectively. TrpM originates from a retained ancient duplication event of a portion of the egtDB gene (latter required for ergothioneine biosynthesis and processively trimethylates L-histidine), and is phylogenetically unrelated to PsiM.

[405] In some embodiments, the TrpM gene from the fungus Psilocybe serbica is introduced into Psilocybe cubensis on an exogenous nucleic acid. The gene comprises SEQ ID NO: 124, which is driven by a GDP promoter, SEQ ID NO: 30. In some embodiments, by expressing the TrpM from the exogenous nucleic acid inside Psilocybe cubensis, DMT is produced. The genetically modified Psilocybe cubensis fungus can further be modified to, e.g., produce increased amounts of L-tryptophan (e.g., introducing an exogenous nucleic acid encoding PsiD), or downregulate enzymatic pathways that use L-tryptophan, to thereby produce greater amounts of DMT. In some embodiments, by expressing the TrpM from the exogenous nucleic acid inside Psilocybe cubensis, DMT is produced. The genetically modified Psilocybe cubensis fungus can further be modified to, e.g., produce increased amounts of L-tryptophan (e.g., introducing an exogenous nucleic acid encoding PsiD), or downregulate enzymatic pathways that use L- tryptophan, to thereby produce greater amounts of DMT. In some embodiments, the TrpM gene comprises a sequence that is at least about: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or is 100% identical to SEQ ID NO: 124.

[406] In some embodiments, a codon optimized Indolethylamine N-methyltransferase from homo sapiens HsINMT (SEQ ID NO: 129) and a codon optimized aromatic L-amino acid decarboxylase (AAAD) from P. cubensis (SEQ ID NO: 122) can be introduced into Psilocybe cubensis. In some embodiments, a genetically modified fungi is crossed with a fungi tryptophan decarboxylase as described above and may be further crossed with a line producing more P- carbolines. AAAD is a noncanonical calcium-activatable aromatic amino acid decarboxylase. AAADs are responsible for alkylamine production in kingdoms of life other than fungi, like L- DOPA decarboxylase which catalyzes the first step in the biosynthesis of monoamine neurotransmitters. AAAD in P. Cubensis shows substrate permissiveness towards L- phenylalanine, L-tyrosine, and L-tryptophan. In Psilocybe mushrooms, L-tryptophan decarboxylation is catalyzed by a neofunctionalized phosphatidylserine decarboxylase-like enzyme (PsiD) rather than by AAAD. In some embodiments, PcAAAD can be used to mediate de novo psilocybin biosynthesis under the control of endogenous calcium signaling and/or elevated environmental calcium concentration. HsINMT (HsINMT, 262 aa) di-methylates tryptamine into DMT.

[407] PsiM catalyzes iterative methyl transfer to the amino group of norbaeocystin to yield psilocybin via a monomethylated intermediate, baeocystin. Psilocybe azurescence is amongst the most potent psilocybin-producing mushrooms. PsiM is regulated on transcriptional level and more copies of PsiM lead to its over expression.

[408] In some embodiments, the genetically modified organism produces an elevated amount of N,N, -dimethyltryptamine in comparison to a naturally occurring otherwise equivalent genetically modified organism. In some embodiments the genetically modified organism expresses a gene product as shown in Table 18. Pharmaceutical Compositions, Nutraceutical Compositions, Supplement Compositions, Formulations and Methods

[409] This disclosure further provides pharmaceutical and/or nutraceutical compositions comprising genetically modified organisms, genetically modified cells, or an extract, a derivative, or product thereof. This disclosure further provides pharmaceutical or nutraceutical reagents, methods of using the same, and methods of making pharmaceutical or nutraceutical compositions comprising genetically modified organisms, genetically modified cells, or an extract, a derivative, or a product thereof.

[410] In some embodiments, a composition comprising a pharmaceutical or nutraceutical composition as disclosed herein can be used for treating or stabilizing conditions or symptoms associated with conditions such as depression, anxiety, post-traumatic stress, addiction or cessation related side-effects such as smoking cessation, and psychological distress including cancer-related psychological distress. In some embodiments, the neurological health condition, disease, or disorder is: a depression, an anxiety, a post-traumatic stress disorder (PTSD), a psychiatric disorder, mental trauma, a mood disorder, a speech disorder, neurodegenerative disease, psychological distress, a compulsion, a compulsive disorder, an obsessive disorder, an expression of a symptom in a neurodivergent individual, cancer-related psychological distress, an addiction, a headache, multiple sclerosis, ameotrophic lateral schlorosis (ALS), Alzheimer’s disease, Parkinson’s disease a phobia, a dementia, a fear, an eating disorder, an ischemic event, or any combination thereof. Specifically, genetically modified organisms described herein, or an extract, a derivative, or product thereof can be used to alleviate various symptoms associated with mental disorders and conditions.

[4H] In some embodiments, compositions comprising the genetically modification organisms described herein can be used to treat particular symptoms. For example, pain, nausea, weight loss, wasting, multiple sclerosis, allergies, infection, vasoconstrictor, depression, migraine, hypertension, post-stroke neuroprotection, as well as inhibition of tumor growth, inhibition of angiogenesis, and inhibition of metastasis, antioxidant, and neuroprotectant. In some embodiments, the genetically modified organisms, can be used to treat persistent muscle spasms, including those that are characteristic of multiple sclerosis, severe arthritis, peripheral neuropathy, intractable pain, migraines, terminal illness requiring end of life care, hydrocephalus with intractable headaches, intractable headache syndromes, neuropathic facial pain, shingles, chronic nonmalignant pain, causalgia, chronic inflammatory demyelinating polyneuropathy, bladder pain, myoclonus, post-concussion syndrome, residual limb pain, obstructive sleep apnea, traumatic brain injury, elevated intraocular pressure, opioids or opiates withdrawal, and/or appetite loss. [412] In some embodiments, compositions comprising the genetically modification organisms described herein can comprise a pharmaceutically or nutraceutically relevant compounds and/or extracts, including flavonoids, monoamine oxidase inhibitors and phytosterols including but not limited to, apigenin, quercetin, cannflavin A, beta-sitosterol, and derivatives and analogues thereof). The compositions of the present disclosure described herein can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like, including those adapted for the following: (1) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (2) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (3) intravaginally or intrarectally, for example, as a pessary, cream or foam; (4) ocularly; (5) transdermally; (6) transmucosally; or (7) nasally.

[413] The pharmaceutical compositions of the disclosure can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[414] The compositions disclosed herein can comprise a preservative, e.g., a compound which can be added to the diluent to essentially reduce bacterial action in the reconstituted formulation, thus facilitating the production of a multi-use reconstituted formulation, for example. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride.

[415] A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.

[416] Compositions described herein may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. In some embodiments, the methods of the disclosure can comprise administration of a composition formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form (e.g., in ampoules or in multi-dose containers) with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[417] Accordingly, this disclosure can provide a pharmaceutical composition comprising an effective amount of a genetically modified organism, a derivative, or an extract thereof, in combination with a pharmaceutically acceptable excipient, carrier, or diluent.

[418] In some embodiments, the genetically modified organism, derivative, or extracts thereof, as disclosed herein, can be used for vaporization, production of e-juice or tincture for e-cigarettes, or for the production of other consumable products such as edibles, balms, or topical spreads. In some embodiments, a modified composition provided herein can be used as a supplement, for example a food supplement. In some embodiments, the genetically modified organisms, or an extract, or a product thereof can be used to make edibles. Edible recipes can begin with the extraction of one or more alkaloids from the organism, which can then be used as an ingredient in various edible recipes. Extraction methods for edibles include extraction into cooking oil, milk, cream, balms, flour and butter. Lipid rich extraction mediums/edibles are believed to facilitate absorption into the blood stream. Lipids may be utilized as excipients in combination with the various compositions provided herein. In other embodiments, compositions provided herein can comprise oral forms, a transdermal forms, an oil formulation, an edible food, or a food substrate, an aqueous dispersion, an emulsion, a solution, a suspension, an elixir, a gel, a syrup, an aerosol, a mist, a powder, a tablet, a lozenge, a gel, a lotion, a paste, a formulated stick, a balm, a cream, or an ointment.

[419] In some embodiments, the genetically modified organism, derivative, or extract thereof, as disclosed herein is a functional mushroom. In some embodiments, the genetically modified organism, derivative, or extract thereof, as disclosed herein is formulated with other functional mushrooms, or extracts, thereof. In some embodiments the genetically modified organism, derivative, or extract thereof, as disclosed herein is formulated with a noortropic herb. In some embodiments the genetically modified organism, derivative, or extract thereof, as disclosed herein is formulated with a phytochemical.

[420] In some embodiments, the compositions described herein further comprise an additional agent selected from at least one of: amyrin, betulinic acid, celastrol, Cesamet (nabilone), marinol (dronabinol; A9-THC), Sativex (cannabidiol; A9-THC), biochanin A, curcumin, cyanidin, desmodianones, delphinidin, (+)-catechin, falcarinol, 18P-Glycyrrhetinic acid, honokiol, isoperrottetin A, kratom, peonidin, pelargonidin, prestimerin, magnolol, malvidin, rutin, 6- methyltetrapterol A, magnolol, miconioside, resveratrol, salvinorin A, yangonin, and 2- arachidonoylgyerol, lysergic acid diethylamide and derivatives and analogues thereof.

[421] In some embodiments, the compositions can be co-administered with an additional agent selected from at least one of: amyrin, betulinic acid, celastrol, Cesamet (nabilone), marinol (dronabinol; A9-THC), Sativex (cannabidiol; A9-THC), biochanin A, curcumin, cyanidin, desmodianones, delphinidin, (+)-catechin, falcarinol, 18P-Glycyrrhetinic acid, honokiol, isoperrottetin A, kratom, peonidin, pelargonidin, prestimerin, magnolol, malvidin, rutin, 6- methyltetrapterol A, magnolol, miconioside, resveratrol, salvinorin A, yangonin, and 2- arachidonoylgyerol, lysergic acid diethylamide and derivatives and analogues thereof.

[422] Provided herein are also kits comprising compositions of the genetically modified cells disclosed herein. The kits can include packaging, instructions, and various compositions provided herein. In some embodiments, the kits can contain additional compositions used to generate the various plants and portions of plants provided herein such as pots, soil, fertilizers, water, and culturing tools.

[423] In some embodiments, a second therapeutic can be administered concurrently, or consecutively, with any composition or pharmaceutical composition described herein.

Manufacturing applications

[424] In some embodiments, the genetically modified organism, derivative, or extracts thereof, as disclosed herein, can be used for the constructing biodegradable plastics.

[425] In some embodiments, the biodegradable is a composite material. In some embodiments, the composite material is used for the construction of an automobile. In some embodiments, the composite material is used for the construction of an aeronautical tool or vessel. In some embodiments, the composite material is used for the construction of tool or vessel in the space industry. In some embodiments, the composite material is used for the construction of garment or textile.

[426] In some embodiments, the genetically engineered organism, derivative, or extract thereof is used as a biodegradable fuel.

EXEMPLARY EMBODIMENTS

[427] While some exemplary embodiments of this disclosure are described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. As a person skilled in the art will readily appreciate, numerous variations, changes, and substitutions are considered within the scope of the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein can be employed in practicing the disclosure. It is intended that the claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[428] In one aspect, this disclosure provides genetically modified organisms that can comprise a genetic modification comprising a heterologous PsiH2 gene that encodes a tryptamine monooxygenase, wherein the tryptamine monooxygenase is expressed in an amount sufficient to produce an alkaloid or a precursor thereof. The heterologous PsiH2 gene can comprise a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical to one of SEQ ID NOS: 15 or 16. The heterologous PsiH2 gene can comprise a sequence that is 99% identical to one of SEQ ID NOS: 15 or 16. The heterologous PsiH2 gene can comprise a sequence that is 100% identical to one of SEQ ID NOS: 15 or 16. The genetically modified organism can be a multicellular organism. The multicellular organism can be from a fungus from division Basidiomycota. In some embodiments, the tryptamine monooxygenase can comprise a tryptamine 4-monooxygenase from one of Psilocybe cyanescens, Psilocybe azurescence, or Psilocybe tampanensis. In some embodiments, the fungus can be from Psilocybe cubensis and the tryptamine monooxygenase may not be naturally expressed at a detectable amount in said fungus. The alkaloid may not be expressed in a detectable amount in a comparable organism without the genetic modification. The alkaloid can be a compound according to formula (I):

-R¹ is H or -CH₃;

-R² is H, -OH, -0P(0)(0M)₂, -C(O)CH₃, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

-R³ is H, -OH, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

-R⁴ and R⁵ are each independently H, -OH, or -OCH₃;

-R^A is H, -COOH; -R^x, R^y, and R^z are each independently H, -CH3, -C(O)CH3, -CH2CH3, or -CH(CH3)2; and -M is H, Li⁺, Na⁺, K⁺ or is absent.

[429] The alkaloid can be a compound according to any one of formulae (la), (lb), and (Ic). The alkaloid can comprise N,N-dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-/.- tryptophan, 5 -hydroxy -/.-tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan-l-aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, or a salt thereof. The alkaloid can be a compound selected from N-acetyl-hydroxytryptamine, 4-hydroxy-Z-tryptophan, 5-hydroxy-/.- tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N-dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium, 4-phosphoryloxy- N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3,4- Methylenedioxymethamphetamine, and derivatives and salts thereof. In some embodiments, the genetic modification can result in increased expression of the alkaloid as compared to a comparable organism without the genetic modification. The alkaloid can comprise psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, or a salt thereof. The alkaloid can be a compound selected from psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, or a salt thereof.

[430] The alkaloid can be a compound according to formula (II):

-X is N, or NH;

- Y is -C-, -CH-, or -CH2-;

-Ring A is a 5-membered heterocyclyl or heteroaryl;

-Ring B is a 6-membered heterocyclyl or heteroaryl;

-R¹ is H or -CH3;

-R² and R³ are each independently selected from H, -OH, and -OCH3;

-R^Ais H, -CH3, -C(O)CH₃, -C(O)OCH₃, -C(O)NH₂, -C(O)OH; and -R^B is absent, H, -CH₃, or -COOH.

[431] The alkaloid can be a compound according to any one of formulae (Ila), (lib), and (lie). The alkaloid can comprise, harmine, harmaline, harmalol, 1,2,3,4-tetrahydroharmine, harmaline, isoharmine, methyl-7-methoxy-beta-carboline-l-l-carboxylate, harmanilic acid, harmanamide, acetylnorharmine, or a salt thereof. In some embodiments, the genetic modification can result in increased expression of the alkaloid as compared to a comparable organism without the genetic modification. The alkaloid can comprise psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, or a salt thereof and further comprise at least one of the compounds selected from harmine, harmaline, harmalol, 1,2,3,4-tetrahydroharmine, harmaline, isoharmine, methyl-7-methoxy-beta-carboline-l- 1 -carboxylate, harmanilic acid, harmanamide, acetylnorharmine, or a derivative or salt thereof.

[432] In some embodiments, the genetically modified organisms described herein can further comprise a second genetic modification that results in increased expression of a helix-loop-helix transcription factor that binds to an E-box motif as compared to a comparable organism without said second genetic modification. The helix-loop-helix transcription factor can be encoded by an exogenous gene that comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to one of SEQ ID NOS: 7 or 14. The second genetic modification can result in upregulated expression of a gene product that can comprise any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, or a psilocybin-related phosphotransferase, as compared to a comparable organism without the second genetic modification. The gene product can be encoded by any one of PsiD, PsiH, PsiK, PsiM, PsiTl, PsiT2, PsiR, or PsiP. In some embodiments, the genetically modified organism can further comprise a heterologous PsiR gene that encodes a helix-loop-helix transcription factor that, when expressed, interacts with a regulatory element of one or more genes, wherein the regulatory element comprises an E-box motif. The one or more genes can comprise any one of PsiD, PsiH, PsiM, PsiTl, PsiT2, or PsiP. In some embodiments, the genetically modified organisms described herein can further comprise an exogenous nucleic acid that results in any one of increased tryptophan decarboxylation, increased tryptamine 4-hydroxylation, increased 4-hydroxytryptamine O-phosphorylation, or increased psilocybin production via sequential N-methylations, as compared to a comparable organism without the exogenous nucleic acid. The genetically modified organism can comprise a fruiting body of a fungus. The genetically modified organism can comprise a non-fruiting body of a fungus. In some embodiments are pharmaceutical compositions that can comprise genetically modified organisms described herein or extracts thereof. The pharmaceutical composition can comprise an effective amount of the genetically modified organism or an extract of the genetically modified organism for treating a health condition. The pharmaceutical compositions can be formulated such that an effective amount of the composition for treatment of the health condition can be delivered in a single dose format. In some embodiments are nutraceutical compositions that can comprise a genetically modified organism described herein or an extract thereof. In some embodiments are supplements that can comprise a genetically modified organism described herein or an extract thereof. In some embodiments are food supplements that can comprise a genetically modified organism described herein or an extract thereof. In some embodiments, the genetic modification can be accomplished by an agrobacterium-mediated insertion of an exogenous sequence into the genetically modified organism. In some embodiments, the genetic modification can be accomplished by contacting a fungal cell with a gene editing system. The gene editing system can comprise one of a CRISPR enzyme, TALE-Nuclease, transposon-based nuclease, Zinc finger nuclease, meganuclease, Mega-TAL or DNA guided nuclease. Also disclosed herein are methods of treatment, wherein the method can comprise administering the genetically modified organism or an extract of the genetically modified organisms described herein to a subject diagnosed with a health condition. The health condition can comprise one of depression, anxiety, post-traumatic stress, addiction, or psychological distress including cancer-related psychological distress. The health condition can be depression, anxiety, post-traumatic stress, addiction, or psychological distress including cancer-related psychological distress.

[433] Also disclosed herein are fungal cells that can comprise a genetic modification that results in the fungal cell expressing a heterologous tryptamine monooxygenase that is not expressed in a detectable amount in a comparable fungal cell without said genetic modification. The fungal cell can be from division Basidiomycota. In some embodiments, the heterologous tryptamine monooxygenase can comprise a tryptamine 4-monooxygenase from one of Psilocybe cyanescens, Psilocybe azurescence, or Psilocybe tampanensis. In some embodiments, the fungus can be from Psilocybe cubensis. In some embodiments, the heterologous tryptamine monooxygenase does not naturally occur at a detectable amount in the fungus. In some embodiments, the fungus can be from Psilocybe cubensis and the heterologous tryptamine monooxygenase does not naturally occur at a detectable amount in the fungus. The heterologous tryptamine monooxygenase can be encoded by a gene comprising a nucleotide sequence that is at that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to any one of SEQ ID NO: 15. The heterologous tryptamine monooxygenase can encode an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to any one of SEQ ID NO: 16. The heterologous tryptamine monooxygenase can be expressed in the fungal cell at an amount sufficient to convert a compound into an alkaloid or a precursor thereof. In some embodiments, the alkaloid can comprise N,N-dimethytryptamine, N-acetyl-hydroxytryptamine, 4- hydroxy-Z-tryptophan, 5 -hydroxy -/.-tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan-l-aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, and derivatives and salts thereof. In some embodiments, the alkaloid can comprise at least one of N,N-dimethytryptamine, N-acetyl- hydroxytryptamine, 4-hydroxy-/.-try ptophan, 5 -hydroxy -/.-tryptophan, 7-hydroxy-Z-try ptophan, 4-phosporyloxy-N,N-dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)- N,N,N-trimethylethan- 1 -aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, and derivatives and salts thereof. In some embodiments, the alkaloid can be a compound selected from N,N- dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-Z-tryptophan, 5-hydroxy-/,- tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N-dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l -aminium, 4-phosphoryloxy- N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3,4- Methylenedioxymethamphetamine, and derivatives and salts thereof. In some embodiments, the expression of the heterologous tryptamine monooxygenase can result in an increased production of the alkaloid as compared to a comparable fungal cell without the genetic modification. The alkaloid can comprise psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N- dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, or a salt thereof. The alkaloid can be a compound selected from psilocybin, psilocin, tryptamine, 4- hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, and derivatives and salts thereof. The expression of the heterologous tryptamine monooxygenase can result in increased production of the alkaloid as compared to a comparable fungal cell without the genetic modification. The alkaloid can comprise psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, and derivatives and salts thereof. The alkaloid can comprise at least one of psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, and derivatives and salts thereof. The alkaloid can be a compound selected from psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, and derivatives and a salt thereof. In some embodiments the fungal cell can further comprise second genetic modification that results in increased expression of a helix-loop-helix transcription factor that binds with an E-box motif as compared to a comparable organism without said second genetic modification. The helix-loop-helix transcription factor can be encoded by a PsiR gene that comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to any one of SEQ ID NOS: 7 or 14. The second genetic modification can result in upregulated expression of any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, or a psilocybin- related phosphotransferase, as compared to a comparable organism without said second genetic modification. The second modification can result in the upregulated expression of a gene product that is encoded by any one of PsiD, PsiH, PsiK, PsiM, PsiTl, PsiT2, PsiR, or PsiP, as compared to a comparable fungal cell without the second genetic modification. The tryptamine monooxygenase can be encoded by an exogenous PsiH2 gene and is expressed in the fungal cell using an exogenous gene promoter, wherein the exogenous gene promoter is GPD. The tryptamine monooxygenase can be encoded by a PsiH2 gene that comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to SEQ ID: 15. The tryptamine monooxygenase can be encoded by a PsiH2 gene that comprises a sequence that is 100% identical to SEQ ID: 15. The fungal cell can be from a fruiting body of a fungus. The fungal cell can be from a non-fruiting body of a fungus. In some embodiments, the fungal cell can further comprise a heterologous PsiR gene that encodes a helix-loop-helix transcription factor that, when expressed, interacts with a E-box motif of one or more genes encoding gene products involved in a biosynthesis pathway of an alkaloid. The one or more genes can be selected from PsiD, PsiH, PsiM, PsiTl, PsiT2, and PsiP. In some embodiments, the fungal cell can further comprise an exogenous nucleic acid that results in any one of increased tryptophan decarboxylation, increased tryptamine 4-hydroxylation, increased 4-hydroxytryptamine O- phosphorylation, or increased psilocybin production via sequential N-methylations, as compared to a comparable fungal cell without the exogenous nucleic acid. The fungal cell can further comprise a second modification that results in an increased expression of a gene product encoded by any one of PsiD, PsiM, PsiH, PsiK, or PsiR, as compared to a comparable fungal cell without the second modification. In some embodiments, are pharmaceutical compositions that can comprise the fungal cells described herein, and extracts thereof. The pharmaceutical composition can comprise an effective amount of the fungal cell or an extract of the fungal cell for treating a health condition. The composition can be formulated such that an effective amount of the composition for treatment of the health condition can be delivered in a single dose format. In some embodiments, are nutraceutical compositions that can comprise the fungal cells described herein and extracts thereof. In some embodiments, are supplement compositions that can comprise the fungal cells described herein and extracts thereof. In some embodiments, are food supplement compositions that can comprise the fungal cells described herein and extracts thereof. In some embodiments, the genetic modification can be accomplished by an agrobacterium-mediated insertion of an exogenous sequence. The genetic modification can be accomplished by contacting a fungal cell with a gene editing system. The gene editing system can comprise any one of a CRISPR enzyme, TALE-Nuclease, transposon-based nuclease, Zinc finger nuclease, meganuclease, Mega-TAL, DNA guided nuclease. In some embodiments are methods of treatment wherein the method can comprise administering the fungal cells or extracts of the fungal cells described herein to a subject diagnosed with a health condition. The health condition can be depression, anxiety, post-traumatic stress, addiction, or psychological distress including cancer- related psychological distress.

[434] In another aspect are genetically modified cells that can comprise a genetic modification comprising an exogenous nucleic acid encoding a helix-loop-helix transcription factor that binds to a regulatory element of one or more genes associated with an alkaloid biosynthesis pathway, wherein the exogenous nucleic acid comprises a sequence that is that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to any one of SEQ ID NO: 7 or 14. The regulatory element can comprise a hexanucleotide sequence that comprises an E-box motif. The one or more genes can comprise PsiD, PsiH, PsiM, PsiTl, PsiT2, or PsiP. PsiP can comprise PsiPl or PsiP2. The genetic modification can result in upregulated expression of any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, or a psilocybin-related phosphotransferase, as compared to a comparable fungal without said second genetic modification. The genetic modification can result in increased expression of a gene produce encoded by any one of PsiD, PsiH, PsiK, PsiTl, PsiT2, or PsiP. PsiP can comprise PsiPl or PsiP2. The genetic modification can result in an increased expression of the helix-loop-helix transcription factor as compared to a comparable fungal cell without the genetic modification. The genetic modification can result in an increased production of an alkaloid as compared to a comparable fungal cell without the genetic modification, wherein the alkaloid is a compound selected from psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, and a salt thereof. The fungal cell can be from division Basidiomycota. The fungal cell can comprise a mycelium. The fungal cell can be from Psilocybe, Conocybe, Gymnopilus, Panaeolus, Pluteus, and Stropharia. The fungal cell can further comprise a second genetic modification that comprises a heterologous PsiH2 gene that encodes a tryptamine monooxygenase. The heterologous PsiH2 gene can comprise a sequence that is 95% identical to any one of SEQ ID NOS: 15-16. The fungal cell can express an alkaloid or a precursor thereof that is not expressed in a detectable amount in a comparable organism without the first and second genetic modifications. The alkaloid can be a compound comprising N,N-dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-/,- tryptophan, 5 -hydroxy -/.-tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan-l-aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, a derivative or a salt thereof. The alkaloid can be a compound selected from N,N-dimethytryptamine, N-acetyl-hydroxytryptamine, 4- hydroxy-Z-tryptophan, 5 -hydroxy -/.-tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan-l-aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, and a salt thereof. In some embodiments, the first and second genetic modifications can result in increased expression of an alkaloid as compared to a comparable fungal cell without the first and second genetic modifications. In some embodiments are pharmaceutical compositions comprising the fungal cells described herein or extracts thereof. The composition can comprise an effective amount of the fungal cell or an extract of the fungal cell for treating a health condition. The composition can be formulated such that an effective amount of the composition for treatment of the health condition can be delivered in a single dose format. In some embodiments are nutraceutical compositions that can comprise the fungal cells described herein or extracts thereof. In some embodiments are supplement compositions that can comprise the fungal cells described herein or extracts thereof. In some embodiments are food supplement compositions that can comprise the fungal cells described herein or extracts thereof. In some embodiments, the genetic modification can be accomplished by contacting a fungal cell with a gene editing system. The genetic modification can be accomplished by an agrobacterium-mediated insertion of the exogenous sequence. The gene editing system can comprise one of a CRISPR enzyme, TALE-Nuclease, transposon-based nuclease, Zinc finger nuclease, meganuclease, Mega-TAL or DNA guided nuclease. In some embodiments are methods of treatment wherein the method can comprise administering a fungal cell described herein or an extract of a fungal cell to a subject diagnosed with a health condition. The health condition can be depression, anxiety, post-traumatic stress, addiction, or psychological distress including cancer- related psychological distress. [435] In another aspect are genetically modified fungal cells that can comprise a first genetic modification that results in an increased expression of a heterologous tryptamine monooxygenase as compared to a comparable fungal cell without the first genetic modification and a second genetic modification that results in an increased expression of a helix-loop-helix transcription factor as compared to a comparable fungal cell without the second genetic modification. The helix-loop- helix transcription factor can bind to a regulatory element of one or more genes that encode products of an alkaloid biosynthesis pathway. The genetically modified fungal cell can be from division Basidiomycota. The heterologous tryptamine monooxygenase can be homologous to a tryptamine monooxygenase that naturally occurs in a fungal cell from one of Psilocybe cyanescens, Psilocybe azurescence, or Psilocybe tampanensis. The heterologous tryptamine monooxygenase can be encoded by a gene comprising a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to one of SEQ ID NOS: 15 or 16. The helix-loop-helix transcription factor can be encoded by a gene that comprises a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to one of SEQ ID NOS: 7 or 14. The regulatory element can comprise a hexanucleotide sequence comprising an E-box motif. The one or more genes can comprise PsiD, PsiH, PsiM, PsiTl, PsiT2, or PsiP. PsiP can comprise PsiPl or PsiP2. The helixloop-helix transcription factor can upregulate expression of the one or more genes as compared to a comparable fungal cell without the second genetic modification. The genetically modification fungal cell can produce an alkaloid that is not naturally expressed at a detectable amount in a comparable fungal cell without the first and second genetic modifications. The genetically modified fungal cell can be from Psilocybe cubensis. The heterologous tryptamine monooxygenase can be encoded by an exogenous nucleic acid. The second genetic modification can result in upregulated expression of any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, or a psilocybin- related phosphotransferase, as compared to a comparable organism without said second genetic modification. In some embodiments, the genetically modified fungal cell can further comprise a modification that results in upregulated expression of at least one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, or a psilocybin-related phosphotransferase. In some embodiments, the first and second genetic modifications can result in an increased production of an alkaloid as compared to a comparable fungal cell without the first and second genetic modifications, wherein the alkaloid is a compound selected from psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, N-acetyl-hydroxytryptamine, 4- hydroxy-Z-tryptophan, 5 -hydroxy-Z-tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan-l-aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, and derivatives and salts thereof. The fungal cell can comprise a mycelium. In some embodiments are pharmaceutical compositions comprising the fungal cells described herein or extracts thereof. The composition can comprise an effective amount of the fungal cell or an extract of the fungal cell for treating a health condition. The composition can be formulated such that an effective amount of the composition for treatment of the health condition can be delivered in a single dose format. In some embodiments are nutraceutical compositions that can comprise the fungal cells described herein or extracts thereof. In some embodiments are supplement compositions that can comprise the fungal cells described herein or extracts thereof. In some embodiments are food supplement compositions that can comprise the fungal cells described herein or extracts thereof. In some embodiments, the first genetic modification or the second genetic modification can be accomplished by contacting a fungal cell with a gene editing system. In some embodiments, the first genetic modification and the second genetic modification can be accomplished by contacting a fungal cell with a gene editing system. The first genetic modification or the second genetic modification can be accomplished by an agrobacterium-mediated insertion of the exogenous sequence. The first genetic modification and the second genetic modification can be accomplished by an agrobacterium-mediated insertion of the exogenous sequence. The gene editing system can comprise one of a CRISPR enzyme, TALE-Nuclease, transposon-based nuclease, Zinc finger nuclease, meganuclease, Mega-TAL or DNA guided nuclease. In some embodiments are methods of treatment wherein the method can comprise administering a fungal cell described herein or an extract of a fungal cell to a subject diagnosed with a health condition. The health condition can be depression, anxiety, post-traumatic stress, addiction, or psychological distress including cancer- related psychological distress.

[436] In another aspect are methods of producing an alkaloid by introducing a genetic modification into a fungal cell, wherein the genetic modification comprises an exogenous nucleic acid that encodes a tryptamine monooxygenase that is heterologous to the fungal cell and expressing the tryptamine monooxygenase in the fungal cell to produce the alkaloid. The tryptamine monooxygenase can be encoded by a PsiH2 gene comprising a nucleotide sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 99% or at least 100% identical to SEQ ID NO: 15 or 16. The exogenous nucleic acid can be introduced into the fungal cell by an agrobacterium. The fungal cell can comprise a mycelium. The fungal cell can be from division Basidiomycota. The heterologous tryptamine monooxygenase is homologous to a tryptamine monooxygenase of a fungus from one of Psilocybe cyanescens, Psilocybe azurescence, or Psilocybe tampanensis. The alkaloid that is produced may not expressed in a detectable amount in a comparable fungus without the genetic modification. The alkaloid can comprise any one of N,N- dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-Z-tryptophan, 5-hydroxy-Z- tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N-dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium, 4-phosphoryloxy- N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3,4- Methylenedioxymethamphetamine, or derivatives and salts thereoffhe alkaloid can be a compound selected from N,N-dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-/.- tryptophan, 5 -hydroxy -/.-tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan-l-aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, and derivatives and salts thereof. In some embodiments, the genetic modification can result in increased expression of the alkaloid as compared to a fungus cell without the genetic modification. The alkaloid can be a compound comprising psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin. The alkaloid can be a compound selected from psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin. In some embodiments, the method can further comprise introducing a second genetic modification into the fungal cell, wherein the second genetic modification results in increased expression of a helix-loop-helix transcription factor that binds to a sequence comprising SEQ ID NO: 7 or 14 as compared to a comparable fungal cell without said second genetic modification. The second genetic modification can be accomplished with a gene editing system. The gene editing system can comprise any one of a CRISPR enzyme, TALE-Nuclease, transposon-based nuclease, Zinc finger nuclease, meganuclease, Mega-TAL or DNA guided nuclease.

[437] In another aspect are methods of producing an alkaloid by introducing a genetic modification into a fungal cell, wherein the genetic modification comprises an exogenous nucleic acid that encodes a tryptamine monooxygenase that is heterologous to the fungal cell and expressing the tryptamine monooxygenase in the fungal cell to produce the alkaloid. In preferred embodiments, the alkaloid produced comprises a compound of Formula (I):

-R¹ is H or -CH₃;

-R³ is H, -OH, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

-R⁴ and R⁵ are each independently H, -OH, or -OCH₃;

-R^A is H, -COOH;

[438] In some embodiments, the tryptamine monooxygenase can be encoded by a PsiH2 gene comprising a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 15 or 16. The exogenous nucleic acid can be introduced into the fungal cell by an agrobacterium. The fungal cell can comprise a mycelium. The fungal cell can be from division Basidiomycota. The heterologous tryptamine monooxygenase is homologous to a tryptamine monooxygenase of a fungus from one of Psilocybe cyanescens, Psilocybe azurescence, or Psilocybe tampanensis. The alkaloid that is produced may not expressed in a detectable amount in a comparable fungus without the genetic modification. In some embodiments, the alkaloid is a compound according to formulae (la), (lb), and (Ic). In some embodiments, the alkaloid is a compound according to:

5 -hydroxy tryptamine (11), 4-hydroxy-tryptophan (12), 4-hydroxy-tryptamine (13), normelatonin (14),

In some embodiments, the alkaloid is a compound selected from compounds 1-19. The alkaloid can be a non-naturally occurring compound. The alkaloid can be an alkaloid known to nature. The alkaloid can be an alkaloid not produced in naturally occurring psilocybin-producing fungi. The alkaloid can be an N-methylated derivative of any one of compounds 1-19. The alkaloid can comprise any one of N-acetyl-hydroxytryptamine, 4-hydroxy-Z-tryptophan, 5-hydroxy-Z- tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N-dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium, 4-phosphoryloxy- N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3,4- Methylenedioxymethamphetamine, and derivatives and salts thereof. The alkaloid can be a compound selected from N,N-dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-/.- tryptophan, 5 -hydroxy -/.-tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan-l-aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, and derivatives and salts thereof. In some embodiments, the genetic modification can result in increased expression of the alkaloid as compared to a fungus cell without the genetic modification. The alkaloid can comprise psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin. The alkaloid can be a compound selected from psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin. In some embodiments, the method can further comprise introducing a second genetic modification into the fungal cell, wherein the second genetic modification results in increased expression of a helix-loop-helix transcription factor that binds to a sequence comprising an E-box motif as compared to a comparable fungal cell without said second genetic modification. In some embodiments, the alkaloid can be formulated in an effective amount for treatment of a health condition. In some embodiments, the alkaloid can be formulated in an effective amount for treatment of a health condition in single dose format. In some embodiments, the alkaloid can be formulated as a pharmaceutical composition in an effective amount for treatment of a health condition. The health condition can be depression, anxiety, post- traumatic stress, addiction, or psychological distress including cancer-related psychological distress. The alkaloid can be formulated in a composition that further comprises a pharmaceutically acceptable carrier. The alkaloid can be formulated for use as a supplement. The alkaloid can be formulated for use as a nutraceutical composition. The alkaloid can be formulated for use as a health supplement. The alkaloid can be formulated for use as a food supplement. The second genetic modification can be accomplished with a gene editing system. The alkaloid can be formulated for any of the uses described herein in an effective amount in single dose format. The gene editing system can comprise any one of a CRISPR enzyme, TALE-Nuclease, transposonbased nuclease, Zinc finger nuclease, meganuclease, Mega-TAL or DNA guided nuclease.

[439] In one aspect, this disclosure provides methods and compositions for making genetically modified organisms with enhanced alkaloid production. The alkaloids can be new to nature alkaloids. The alkaloids can be new to the organism from which the alkaloids are produced. The alkaloids can be alkaloids that are rare. [440] In some embodiments, this disclosure involves introducing a heterologous nucleic acid into a fungal organism, wherein the expression of the heterologous nucleic acid leads to the production of a new to nature alkaloid. In some embodiments, nucleic acid encodes a TrpM gene from the fungus Psilocybe serbica. In some embodiments, the TrpM gene is introduced into Psilocybe cubensis on an exogenous nucleic acid. The gene can comprises a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 124. Expression of the gene can be driven by a GDP promoter, e.g., SEQ ID NO: 30. By expressing the TrpM from the exogenous nucleic acid inside Psilocybe cubensis one or more alkaloids can be produced. In some embodiments, the one or more alkaloids are new to the organism from which they are produced. In some embodiments, the one or more alkaloids produced includes DMT.

[441] In some embodiments, this disclosure involves introducing a heterologous nucleic acid into a fungal organism, wherein the expression of the heterologous nucleic acid leads to the production of a new to nature alkaloid. In some embodiments, the nucleic acid encodes a PsiM gene from P. Azurescence, which has been described as being more efficient in producing psilocybin. Thus, in some embodiments wherein the gene is introduced into Psilocybe cubensis, the genetic modification can result in increased production of psilocybin. The gene can comprise a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 121. Expression of the gene can be driven by a GDP promoter, e.g., SEQ ID NO: 30.

[442] In some embodiments, as discussed below, a nucleic acid encoding an indolethylamine N- methyltransferase gene from homo sapiens HsINMT can be incorporate into an organism. The gene can comprise a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 129. In some embodiments, a nucleic acid encoding an aromatic L-amino acid decarboxylase (AAAD) from P. cubensis can be incorporated into an organism. The gene can comprise a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 122. In some embodiments, a gene encoding strictosidine synthase (STST) from Catharanthus roseus, can be incorporated into an organism. The gene can comprise a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 125. In some embodiments, a McbB gene comprising a sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 126 can be incorporated into an organism.

[443] In some embodiments, PsiD gene over-expression comprises a vector expressing PsiD gene under the control of a 35S promoter (Table 26: SEQ ID NO: 304, 17,647bp). In some embodiments, PsiH gene over-expression comprises a vector expressing PsiH gene under the control of a 35S promoter (Table 26: SEQ ID NO: 303, 18,494bp). In some embodiments, PsiK gene over-expression comprises a vector expressing PsiK gene under the control of a 35 S promoter (Table 26: SEQ ID NO: 552, 17,420bp). In some embodiments, PsiM gene over-expression comprises a vector expressing PsiM gene under the control of a 35S promoter (Table 26: SEQ ID NO: 551, 17,267bp). In some embodiments, PsiR gene over-expression comprises a vector expressing PsiR gene under the control of a GPD promoter (Table 26: SEQ ID NO: 308). In some embodiments, PsiH2 gene over-expression comprises a vector expressing PsiH2 gene under the control of a GPD promoter (Table 26: SEQ ID NO: 309 and SEQ ID NO: 310).

[444] In some embodiments, Psi genes over-expression comprises a vector expressing Psi genes under the control of a GcDEDl promoter (Table 26: SEQ ID NO: 305, 9,462bp). In some embodiments, Psi genes over-expression comprises a vector expressing Psi genes under the control of a GPD promoter (Table 26: SEQ ID NO: 106, 8,067bp).

[445] In some embodiments, PsiD over-expression comprises a vector expressing Psi genes under the control of a GPD promoter (Table 26: SEQ ID NO: 307), resulting in a fungus comprising a blue phenotype.

TABLE 26. Psilocybin expression vector sequences.

[446] In some embodiments, genetically modifying an organism involves introducing an exogenous nucleic acid into the organism. In some embodiments, the exogenous nucleic acid encodes PsiM. In some embodiments, the PsiM gene is codon optimized. In some embodiments, the codon optimized PsiM is driven by a GPD promoter. In some embodiments, the exogenous nucleic acid comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 500.

[447] In some embodiments, the exogenous nucleic acid encodes an aromatic L-amino acid decarboxylase (AAAD) gene from P.cubensis. In some embodiments, the AAAD is codon optimized for expression in P. cubensis. In some embodiments, the AAAD gene is driven by a GPD promoter. In some embodiments, the exogenous nucleic acid comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 501.

[448] In some embodiments, the exogenous nucleic acid encodes a heterologous protein. In some embodiments, the nucleic acid encodes a PsiM gene from P. Azurescence. In some embodiments, the PsiM gene is codon optimized for expression in P. cubensis. In some embodiments, the codon optimized PsiM is driven by a GPD promoter. In some embodiments, the exogenous nucleic acid comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 502.

[449] In some embodiments, the exogenous nucleic acid encodes a heterologous protein. In some embodiments, the heterologous protein comprises TrpM from P. serbica. In some embodiments, the TrpM gene is codon optimized for expression in P. cubensis. In some embodiments, the TrpM gene is driven by a GPD promoter. In some embodiments, the exogenous nucleic acid comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 503.

[450] In some embodiments, the exogenous nucleic acid encodes a strictosidine synthase gene (STST) from Catharanthus roseus. In some embodiments, the STST gene is codon optimized for expression in P. cubensis. In some embodiments, the STST gene is driven by a GPD promoter. In some embodiments, the exogenous nucleic acid comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 504.

[451] In some embodiments, the exogenous nucleic acid encodes an indolethylamine N- methyltransferase (INMT) gene from homo sapiens. In some embodiments, the INMT gene is codon optimized for expression in P. cubensis. In some embodiments, the INMT gene is driven by a GPD promoter. In some embodiments, the exogenous nucleic acid comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 505.

[452] In some embodiments, the exogenous nucleic acid encodes McbB from marine actinomycete M. thermotolerans. In some embodiments, the MccB gene is codon optimized for expression in P. cubensis. In some embodiments, the MccB gene is driven by a GPD promoter. In some embodiments, the exogenous nucleic acid comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 506.

[453] In some embodiments, this disclosure provides tools and reagents for making a genetic modification. In some embodiments, this disclosure provides vectors for introducing guide nucleic acids into an organism. In some embodiments, the vector comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 507.

[454] In some embodiments, the vector comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 508. In some embodiments, the vector comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to a plasmid listed in Table 27.

[455] In some embodiments, the vector comprises a PsiH2 gene and comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 509.

In some embodiments, the vector comprises a PsiH2 gene and comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 510. In some embodiments, the vector comprises a PsiR gene comprises a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical to SEQ ID NO: 511.

[456] Table 27. Exemplary Plasmid sequences

[457] In some embodiments, this disclosure provides reagents, such as plasmids, useful for introducing genetic modifications into an organism. In some embodiments, the plasmids are optimized for introducing a genetic modification into the genome of a fungal cell. In some embodiments, the plasmids encode a gene editing system. In some embodiments, the plasmids encode one or more guide polynucleotides separately, or in combination with a gene editing system. In some embodiments, the guide polynucleotide comprises a targeting sequence for binding to a psilocybin synthase gene to thereby introduce a genetic modification into the psilocybin synthase gene. In some embodiments, the psilocybin synthase gene comprises PsiPl. In some embodiments, the psilocybin synthase gene comprises TrpE. In some embodiments, the guide polynucleotide comprises a sequence for binding to a non-coding region in a psi locus. [174] For example, provided herein are plasmids comprising guide polynucleotides with targeting sequences for binding PsiPl and PsiP2 in combination with a codon optimized Cas9 and hygromycin resistance gene. See SEQ ID NOS: 601-602. Provided herein are also plasmids comprising guide polynucleotides for binding TrpE and watermark (i.e., a non-coding region in Psi locus) sequences in combination with a codon optimized Cas9 and hygromycin resistance (see SEQ ID NO: 603). Provided herein are plasmids encoding guide polynucleotides comprising targeting sequences for watermark (i.e., a non-coding region in Psi locus) sequences in combination with a codon optimized Cas9 and hygromycin resistance (see SEQ ID NO: 604). Provided herein are also plasmids comprising guide polynucleotides with target sequences for TrpE in combination with a codon optimized Cas9 and hygromycin resistance (see SEQ ID NO: 605). Provided herein are also plasmids encoding guide polynucleotides comprising sequences for binding PsiR in combination with a codon optimized Cas9 and hygromycin resistance (See SEQ ID NO: 606). In some embodiments, the plasmid is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to one of the plasmids listed in TABLE 28.

TABLE 28. Exemplary gene editing plasmids

[458] A genetic modification can involve the upregulated expression of PsiR. An analysis of PsiD, PsiM, PsiT2, PsiH, PsiK, PsiTl, PsiP, and PsiL reveals that PsiD, PsiH, PsiM, PsiT2 genes contain one E-box motif in their promoters (PsiTl has two), whereas PsiP contains 4 E-box motifs (500bp upstream of ATG). PsiL and PsiK do not have E-box motif, or E-box motifs in their upstream regions. Thus, upregulating PsiR is expected to modulate expression of the Psi genes thereby enhancing production of one or more alkaloids.

[459] Listed below in TABLE 29 are regulatory sequences of PsiD, PsiM, PsiT2, PsiH, PsiK, PsiTl, PsiP, and PsiL:

TABLE 29. Psilocybe cubensis Regulatory Gene Sequences

[460] In silico analysis of PsiR expression levels and splice forms was performed using a publicly available database.

[461L Primers used to validate PsiR splice varians were as follows :

[462] Transformation has been used to introduce gene editing systems into some fungal species. As in plants, certain steps for introducing a gene editing system into a fungal cell can include preparation of plasmids or ribonucleoproteins, delivery of said plasmids or ribonucleoproteins, protoplast regeneration, and mutant identification. However, fungal protoplasts are more than twice as small those of plants. Therefore, much fewer plasmids or ribonucleoproteins may be adsorbed onto a surface of fungal protoplasts compared to those of plants. Due to the low ratio of plasmids or ribonucleoproteins absorbed onto surface of fungal cells, gene editing efficiencies can be reduced. In some embodiments, to improve transformation efficiency, a reagent is added. In some embodiments, the reagent comprises a detergent or a nonionic surfactant that impacts permeability of the fungal protoplast cell wall. In some embodiments, the reagent is Triton X-100. In some embodiments, the reagent comprises a lipid particle that can encapsulate the endonuclease and the guide polynucleotide, or the one or more nucleic acids encoding said endonuclease and the guide polynucleotide to thereby deliver the gene editing system into the fungal cell. The presence of the reagent can increase transformation efficiency by about 3-fold, about 4-fold, about 5-fold, or more.

[463] In some embodiments, the reagent can be a compound that impacts cell division. In some embodiments, the reagent can be a reagent that acts by depolymerizing microtubules. In some embodiments, the reagent can be a compound that increases the formation of monokaryotic protoplasts, which can greatly improve the efficiency of obtaining homozygous transformants and eliminate multi-round single-spore isolation process in the late stage of transformation. In some embodiments, the reagent comprises inositol. In some embodiments, the reagent comprises benomyl.

[464] In some embodiments, the gene editing system comprises the one or more nucleic acids encoding the endonuclease and the at least one guide polynucleotide. In some embodiments, the endonuclease and the at least one guide polynucleotide are encoded on a single nucleic acid. In some embodiments, the endonuclease and the at least one guide polynucleotide are encoded on different nucleic acids. In some embodiments, the one or more nucleic acids comprises one or more nucleic acids that are non-replicating such that after the gene editing system is introduced into the fungal cell, the gene editing system is not passed down to prodigy cells during cell division. Thus, negative downstream effects of the gene editing system can be reduced or eliminated. As such, the genome editing system may be widely applicable to construction of genome-edited fungi for food or medicinal purposes.

[465] In some embodiments, the one or more nucleic acids comprises a gene promoter that drives expression of the endonuclease or the at least one guide polynucleotide in the fungal cell. In some embodiments, the promoter is a fungal promoter. In some embodiments, the gene promoter is a U6 gene promoter. In some embodiments, the gene promoter is a GDP gene promoter. In some embodiments, the gene promoter is a U6 promoter that has a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% identical to any one of SEQ ID NOS: 400-403. In some embodiments, the gene promoter is a GDP promoter that has a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% identical of SEQ ID NO: 406. In some embodiments, the one or more nucleic acids comprise a first gene promoter that drives expression of the endonuclease and a second gene promoter that drives expression of the at least one guide polynucleotide, wherein the first gene protomer is different from the second gene promoter. For example, in some embodiments, the first gene promoter is a GDP gene promoter, and the second gene promoter is a U6 gene promoter.

[466] In some embodiments, the endonuclease is in a complex with the at least one guide polynucleotide. In some embodiments, the endonuclease and the at least one polynucleotide are delivered as an active ribonucleoprotein. In some embodiments, the at least one guide polynucleotide comprises a sequence that can hybridize to one of the genes listed in Tables 1-2, 3A, or 3B. In some embodiments, the at least one guide polynucleotide binds to the gene within 100 bases of the sequence comprising one of SEQ ID NOS: 60-116. In some embodiments, the at least one guide polynucleotide binds to the gene at least partially overlapping the sequence comprising one of SEQ ID NOS: 60-116. In some embodiments, the at least one guide polynucleotide comprises a targeting sequence that is complementary to one of SEQ ID NOS: 60-116. In some embodiments, the at least one guide polynucleotide comprises a targeting sequence that binds to one of SEQ ID NOS: 60-116.

[467] In some embodiments, upon binding of the gene editing system to the target, the gene editing system introduces a genetic modification that results in the downregulation of the targeted gene. For example, in some embodiments, the activity of the gene editing system on the gene results in a deleterious frameshift mutation that eliminates expression of a functional gene product. The deleterious frameshift mutation can be introduced by, for example, adding or removing a single nucleotide.

[468] In some embodiments, the at least one guide polynucleotide comprises a sequence that can bind to a sequence that is at least 75 percent, 80 percent, 90 percent or 95 percent identical to one of SEQ ID NOS: 60-116. In some embodiments, the at least one guide polynucleotide can bind under highly stringent conditions. Highly stringent conditions can comprise a stringent wash under high temperature, e.g., about 60 degrees to about 65 degrees Celsius, and low salt, e.g., about 0.01 M. In some embodiments, the at least one guide polynucleotide comprises a sequence that has at least 75 percent, 80 percent, 90 percent or 95 percent identity to one of SEQ ID NOS: 60-116.

[469] In some embodiments, the gene editing system comprises multiplex capabilities. Accordingly, in some embodiments, the gene editing comprises at least two guide polynucleotides.

[470] In some embodiments, the gene editing system comprises a regent that facilities the introduction of the gene editing system into the fungal cell. In some embodiments, the reagent comprises a detergent. In some embodiments, the reagent comprises a nonionic surfactant. In some embodiments, the reagent is Triton X-100. A nonionic surfactant, e.g., Triton X-100, can be useful to increase permeability of the fungal cell to accept the gene editing system. The regent can be provided at a concentration of, for example, 0.10 - 0.25 mM, such as, 0.20 mM. In some embodiments, the fungal cell is incubated with the gene editing system in the presence of 0.20 mM of Triton X-100 for about 1 hour at room temperature.

[471] In some embodiments, the reagent comprises a lipid nanoparticle. Surprisingly, the lipid nanoparticle can encapsulate and deliver a Cas endonuclease guide RNA complex into a fungal cell such as a fungal protoplast. Advantageously, by delivering the gene editing system as an active protein, the gene editing system cannot be carried down to subsequent cell generations. The gene editing system can comprise the endonuclease and the at least one guide polynucleotide in one or more compositions. In some embodiments, the endonuclease and the at least one guide polynucleotide are provided in the same composition. In some embodiments, the endonuclease and the guide polynucleotide are delivered in separate compositions. In methods of use, the endonuclease and the at least one polynucleotide can be delivered sequentially or concurrently.

[472] In some embodiments, the engineered fungus comprises a concentration of psilocybin that is at least 10% greater than a concentration of psilocybin in a comparable fungus devoid of said genetic modification. The engineered fungus can be selected from the group consisting of Psilocybe, Conocybe, Gyranopilus, Panaeolus, Pluteus, and Stropharia. In some instances, the engineered fungus includes one or more transgenes are selected from the group consisting of (i) PsiD, (ii) PsiD and PsiK, (iii) PsiD, PsiK, PsiM, and (iv) PsiD, PsiK, PsiM, PsiH.

[473] Disclosed herein is a method for enhanced alkaloid production comprising obtaining a mycelial mass comprising a population of fungal cells, wherein the population of fungal cells comprises a genetic modification that results in a population of genetically modified fungal cells producing an increased amount of an alkaloid as compared to a comparable wild-type population of fungal cells.

ADDITIONAL EXEMPLARY EMBODIMENTS

1. An engineered fungal cell comprising a heterologous PsiL gene.

2. The engineered fungal cell of embodiment 1, wherein the heterologous PsiL gene comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 322.

3. The engineered fungal cell of embodiment 1, wherein the heterologous PsiL gene comprises a sequence that has 100% identity to SEQ ID NO: 322.

4. The engineered fungal cell of embodiment 1, that is comprised in a multicellular organism.

5. The multicellular organism of embodiment 4, that is from division Basidiomycota.

6. The multicellular organism of embodiment 5, that is a species selected from the group consisting of: Psilocybe cyanescens, Psilocybe azurescence, and Psilocybe tampanensis.

7. The multicellular organism of embodiment 6, that is the species Psilocybe cubensis.

8. The engineered fungal cell of embodiment 1, that comprises an alkaloid.

9. The engineered fungal cell of embodiment 8, wherein the alkaloid is selected from the group consisting of: N,N-dimethyltryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-/,- tryptophan, 5 -hydroxy -/.-tryptophan, 7-hydroxy-Z-tryptophan, isonorbaeocystin, 4- phosporyloxy-N,N-dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol- 3-yl)-N,N,N-trimethylethan-l-aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, harmine, harmane, a P-carboline, and a salt of any of these. The engineered fungal cell of embodiment 1, which has an increased expression of an alkaloid as compared to a comparable fungal cell without a heterologous PsiL gene. A method of overexpressing a gene involved in psilocybin biosynthesis in a psilocybe protoplast, the method comprising: i) targeting a gene involved in a psilocybin biosynthesis through homologous directed repair, wherein a self-replicating plasmid is transfected into a psilocybe protoplast; and ii) introducing a polynucleotide comprising a B-AMA1 replication origin sequence into the self-replicating plasmid in the psilocybe protoplast, wherein the self-replicating plasmid is not stably integrated into the genome of the protoplast; thereby overexpressing the gene in the psilocybe protoplast. The method of 11, wherein the psilocybe protoplast is a psilocybe cubensis protoplast. The method of embodiment 11, wherein the gene involved in psilocybin biosynthesis is a PsiD gene. The method of embodiment 12, wherein the PsiD gene has at least 95% identity to SEQ ID NO: 120. The method of embodiment 11, wherein the self-replicating plasmid comprises a cassette comprising a hygromycin resistance gene. The method of embodiment 15, wherein the hygromycin resistance gene is subsequently spontaneously removed from the psilocybe protoplast without selection pressure. The method of embodiment 11, wherein the self-replicating plasmid comprising the polynucleotide comprising the B-AMA1 sequence is comprised in an spCas sequence. The method of embodiment 17, wherein the polynucleotide comprising B-AMA1 replication origin sequence comprises at least 95% identity to any one of SEQ ID NOs: 640-647. The method of embodiment 11, wherein the self-replicating plasmid comprising the polynucleotide comprising the B-AMA1 replication origin sequence comprises at least 95% identity to SEQ ID NO: 648 or 649. EXAMPLES

Example 1. Gene diversity driven compound generation.

[474] Phylogenetic analysis ofPsiH: A phylogenetic analysis of PsiH was performed to evaluate the relatedness ofPsiH genes from four species of psilocybin-producing fungi: Psilocybe cubensis, Psilocybe azurescens, Psilocybe cyanescens, and Psilocybe tampanensis. Amino acid sequences generated by PsiH and/or PsiH2 from the four fungal species were retrieved from a public genome database, the National Center for Biotechnology Information (NCBI). For Psilocybe cubensis genome, a publicly available sequence dataset for Psilocybe cubensis from the “P.envy” genome (available on NCBI at https://www.ncbi.nlm.nih.gOv/assembly/GCA_017499595.l) was used. For Psilocybe tampanensis, Psilocybe cyanescens and Psilocybe azurescens, a de novo assembly was required (https://www.ncbi. nlm.nih.gov/genome/?term=psilocybe). See FIG. 4 for PsiH and PsiH2 sequences that were aligned and compared for analysis.

[475] FIG. 4 shows a comparison ofPsiH and PsiH2 gene products from four different psilocybin-producing fungi. A comparison of the aligned sequences reveals Psilocybe cubensis, Psilocybe azurescens, Psilocybe cyanescens, and Psilocybe tampanensis produce different amino acid polypeptides. These data show different psilocybin-producing fungi produce different amino acid polypeptides which suggested fungi can host different alkaloid biosynthesis pathways that may produce different alkaloids.

[476] FIG. 5 shows a phylogenetic tree generated for PsiH and PsiH2 genes from four psilocybin producing fungi. In particular, the phylogenetic tree illustrates the relatedness ofPsiH and PsiH2 genes from fungal species Psilocybe cubensis, Psilocybe azurescens, Psilocybe cyanescens, and Psilocybe tampanensis. The branch points along the tree indicate relatedness of the genes. The analysis revealed that Psilocybe cubensis does not possess PsiH2 in its genome and led to the exciting possibility that transgenic PsiH2 fungi in a fungal cell from Psilocybe cubensis may produce new alkaloids.

Example 2. Generating transgenic fungi

[477] Fungi from Psilocybe cubensis was genetically modified to overexpress tryptophan decarboxylase to demonstrate the ability to produce transgenic fungi. The methods are described below.

[478] Preparation of fungal material'. Fungal material was prepared for transformation with one of two plasmids encoding PsiD. The plasmids included pCambial300:GPDstart_intron_6bp:Gus no intron:stop:polyA (GT 4) (SEQ ID NO: 308)., and pCambial300:GPD:intron-PsiD-stop:polyA (GT 6) (SEQ ID NO: 307). The plasmids include a PsiD gene with SEQ ID NO. 120, driven by a promoter having SEQ ID NO. 30.

[479] FIG. 6 shows a plasmid encoding PsiD. In particular, illustrated is the GT6 plasmid.

[480] Non-transgenic fungal cells from Psilocybe cubensis mycelia were maintained on potato dextrose agar (PDA) at 25 Celsius in the dark. The mycelia (cells 3 weeks old or less) were cut into small blocks from agar plates and added to 100 mL potato dextrose broth (PDB) media. The mycelium cultures were incubated in a shaker incubator (at 175 rpm) for six (6) days in the dark at 28 Celsius.

[481] Following incubation, six (6) day old Psilocybe cubensis were transferred to fresh PDB medium and homogenized using an Ultra-Turrax homogenizer 24 hours before inoculation. Hyphal fragments were transferred to fresh PDB and grown for 24 hours to give a uniform mycelial slurry under same conditions as the originally maintained mycelia sample.

[482] Bacteria preparation '. A. tumefaciens strains LBA4404/AGL1 carrying a plasmid of GT4 or GT6 were grown for 24-48 hours in Lysogeny broth (LB) medium supplemented with appropriate antibiotics prior to inoculation. On the day of inoculation, bacterial cultures were diluted to an optical density of 0.15 at 660 nm with agrobacterium induction medium (AIM) (Induction medium (AIM) [MM containing 0.5% (w/v) glycerol, 0.2 mM acetosyringone (AS), 40 mM 2-N-morpholino-ethane sulfonic acid (MES), pH 5.3) and grown for an additional 5-6 hours in a shaker incubator at 28 Celsius.

[483] Agrobacterium-mediated transformation'. A 25 mL aliquot of the mycelial suspension (uniform mycelial slurry) was mixed with 25 mL of one of the bacterial cultures comprising GT4 as prepared as disclosed above. A second 25 mL aliquot of the mycelial suspension (uniform mycelial slurry) was mixed with 25 mL of one of the bacterial cultures comprising GT6 as prepared as disclosed above. 1 mL of each of the mixtures was spread on cellophane discs following vacuum treatment and overlaid on agar plates and incubated at 25 Celsius for 72-92 hours in the dark. Agrobacterium contamination level was observed after 72 hours. Cocultivation was stopped if agrobacterium was visible, otherwise, incubation was continued up to 92 hours.

[484] After cocultivation, cellophane discs were transferred to Selection 1, which included PDA medium containing 200 mg/L Timentin to kill residual agrobacterium cells and 80 mg/L Hygromycin to select fungal transformants. Selection 1 was incubated for 10-15 days at 25 Celsius. After Selection 1, individual colonies were transferred to PDA medium containing 80 mg/L Hygromycin and 200 mg/L Timentin (Selection 2). Example 3. Confirmation of PsiD transgenic fungi

[485] Transformed PsiD colonies generated according to Example 2 demonstrated significant growth and were transferred to new selection media every 15 days to selectively grow cells with the exogenous nucleic acid integrated. To confirm integration of the exogenous nucleic acid encoding tryptophan decarboxylase, a GUS assay, short for “beta-glucuronidase”, and PCR analysis were performed. Both assays confirmed transformation of the exogenous nucleic acid.

[486] FIG. 7 shows a PCR gel confirming the fungal cell was genetically modified. In particular, the PCR gel confirms incorporation of the GT6 plasmid into fungal mycelia. “C+” indicates a lane loaded with a positive control. “C-” indicates a lane loaded with a negative control, “wt” indicates a lane from a PCR run performed on wild-type fungal material. The absence of signal in the “wt” lane and positive signal at -about 500 bp in lanes 1-16 is indicative that the PsiD transgene is integrated into the genome of the fungal mycelia.

[487] After confirming integration of the exogenous nucleic acid, additional PCR assays were performed to assess whether tryptophan decarboxylase expression (encoded by PsiD) was elevated. Specifically, quantitative real-time PCR (RT-PCR) assays were performed to assess levels of mRNA encoded by PsiD.

[488] FIG. 8 shows RT-PCR gels that confirm PsiD upregulation in transgenic mycelia. The gels are from cDNA analyses of PsiD mRNA expression in mycelia transformed with the GT6 plasmid. Specifically, the top gel 801 is a gel from a cDNA analysis (RT-PCR) of total expressed mRNA in transgenic mycelia. The data show clear upregulated expression of the transgenic mycelia (bands in lanes 1-16 at about 500 bp (805)) as compared to comparable wild-type mycelia devoid of the genetic modification, i.e., the PsiD transgene. As shown, the expression of PsiD in the transgenic mycelia is approximately 6-10-fold higher than the basal level of expression seen in the wild type cells. The lower gel 803 is a cDNA analysis of expressed PsiD mRNA in transgenic mycelia in which no reverse transcriptase (thus no mRNA is converted into cDNA) was added during the RT-PCR assay confirming the PsiD observed in the top gel 801 is expressed mRNA and not contaminating DNA.

Example 4. Transgenic fungi expressing elevated levels of tryptophan decarboxylase demonstrate an altered phenotype

[489] The transgenic and non-transgenic colonies were cultured on selection plates with Hygromycin (80 mL) and Timentin (200 mg/L). Surprisingly the transgenic mycelia exhibited a phenotype that was visually distinct from the phenotype of non-transgenic colonies. In particular, the mycelial mass from the transgenic fungus showed a blue coloration that was not apparent in non-genetically modified fungi.

[490] FIG. 9 shows non-transgenic wild-type mycelia and transgenic mycelia that express elevated levels of PsiD. In particular, the figure shows selection plates of non-transgenic wildtype mycelia 901 and transgenic mycelia 903 that express elevated levels of PsiD. As demonstrated, the transgenic mycelia 903 exhibit a phenotype that is visually district from the comparable wild-type mycelia of the non-transgenic wild-type mycelia 901. The phenotype comprises a blue coloration that is district from the wild-type mycelia 901. The blue coloration of the transgenic mycelia 903 is illustrated in the black and white image as a different gradient of grey compared to the wild-type mycelia 901. The blue coloration is believed to be indicative of a higher concentration of psilocybin in the transgenic fungi.

[491] The mycelia were grown into mycelial masses from which primordia were produced. In particular, genetically modified mycelia were crossed with wildtype (e.g., GT4) in cups containing casing. Each was wrapped in aluminum foil and placed in the dark at 27 Celsius for incubation. After 18 days, the cakes were transferred into bigger pots with vents open. For one set of the cakes, the pots were kept in the cup with wet casing, and the second set was removed from their cups with the casing on top. No pin head of the fruiting body was observed over the casing in those still inside the pots.

[492] FIG. 10 shows a transgenic mycelial mass 1004 upon primordia formation 1005. As discussed above, the transgenic mycelial mass 1004 has a blue coloration that is distinct from a wild-type mycelia mass 1003, a comer of which is illustrated in the upper left comer of the figure. The blue coloration is shown in the black and white image as a darker gradient of grey.

[493] Dissection of the fruiting bodies and extended exposure to air produced the phenotypic tissue expression of the genetically modified sample in comparison to the unmodified fruiting body.

[494] FIG. 11 shows a side-by-side comparison of a PsiD transgenic fungus 1103 compared to a wild-type fungus 1105. The PsiD transgenic fungus comprises a blue coloration that is visibly distinct from the wild-type fungus 1105. The blue coloration is suggestive of an increased quantity of psilocin in the transgenic fungus 1103 as compared to the wild-type fungus 1105. Since psilocin is derived from psilocybin, the transgenic fungus’ phenotype (i.e., the blue coloration) suggests that the transgenic fungus has an increased amount of psilocybin as compared to the wild-type fungus 1105.

Example 5. Alkaloidal content of PsiD transgenic fungi [495] The psilocybin content of the genetically modified mushrooms was analyzed by liquid chromatography/mass spectrometry to determine amounts of alkaloids present in the transgenic fungi. Liquid chromatography-mass spectrometry (LC-MS) is an analytical method that combines the features of liquid-chromatography and mass spectrometry to identify different substances within a test sample. To assess the alkaloids, present in transgenic fungi, LC-MS analyses were conducted at two independent facilities.

[496] Briefly, fruiting bodies of transgenic fungi and non-transgenic fungi were dissected and transferred to 50 mL falcon tubes and snap frozen in liquid nitrogen. Wet weight was measured and samples were maintained at -80 degrees Celsius. Samples were desiccated in a freeze drier at -45 degrees Celsius at 0.05 bar for 24 hours. The freeze-dried samples were then ground to a fine powder at room temperature using a mortar and pestle. Ground samples were transferred to a 50 mL tube and dry weight was measured. 7.1 grams of dry ground sample were transferred to a subsequent set of 50 mL tubes and sent for analysis.

[497] FIG. 12 shows a biosynthesis pathway of alkaloids downstream from PsiD that were identified as upregulated in the transgenic fungus as compared with a wild-type fungus devoid of a genetic modification. The upregulated alkaloids included tryptamine, 4-hydroxytryptamine, norbaeocystin, baeocystin, norpsilocin, psilocybin, and psilocin.

[498] FIG. 13 shows graphs of concentrations of alkaloids measured in PsiD transgenic and wild-type fungi. As illustrated, the data show alkaloids norbaeocystin, baeocystin, norpsilocin, psilocybin, and psilocin, are substantially upregulated as compared with a comparable wild-type fungus. Specifically, norbaeocystin is 34.7-53.7 times upregulated as compared with a comparable wild-type fungus, baeocystin is 17.5-12.5 times upregulated as compared with a comparable wild-type fungus, norpsilocin is 10-12.1 times upregulated as compared with a comparable wild-type fungus, psilocybin is 2.2-2.3 times upregulated as compared with a comparable wild-type fungus, and psilocin is 5.3-5.4 times upregulated in the PsiD transgenic fungi as compared to a comparable wild-type fungus as compared with a comparable wild-type fungus. For psilocybin and psilocin, concentrations in fungi are reported assuming a 100% recovery in ppm (pg/g). For norpsilocin, baesocystin, and norbaeocystin, data are reported as area counts detected by the LC-MS/MS. Compounds were subjected to confirmation with standards.

[499] FIG. 14 shows the content of psilocybin and psilocin in PsiD transgenic fungi as compared with wild-type fungi.

[500] FIG. 15 shows amounts of certain alkaloids measured in transgenic and wild-type fungi by LC-MS. As illustrated, the data confirm prior results demonstrating norbaeocystin, baecystin, norpsilocin, psilocybin, and psilocin, are present at a substantially higher amount as compared with a comparable wild-type fungus.

[501] FIG. 16 shows the content of psilocybin and psilocin in the PsiD transgenic fungi as compared with wild-type fungi. The data confirm that the genetic modification of the PsiD transgenic fungus results in at least a 4.2-fold increase in production of psilocybin and at least a 2.5-fold increase in production of psilocin as compared to a comparable wild-type fungus.

[502] FIG. 17 illustrates alkaloids formed from psilocin. In particular, illustrated are certain alkaloids that can be formed by the oxidation of psilocin. The illustrated alkaloids include quinonoid and quinoid dimers.

[503] FIGS. 18A and 18B are LC-MS data on quinoid and quinoid dimers as compared with psilocin from three different samples. The three samples include transgenic PsiD fungi (Nos. 1771 and 1772), and a wild type control (No. 1773). FIG. 18A reports peak areas of quinoid and quinoid dimers that enzymatically produced. FIG. 18B reports peak areas from electrospray ionization (ESI) produced product. The ESI produced quinoid and quinoid dimers are psilocin concentration dependent.

[504] FIGS. 19A-19D shows relative amounts of alkaloids in PsiD transgenic fungi as compared with wild-type fungi. In particular, the data show relative amounts of 4- hydroxytryptamine, 4-hydroxytrimethyltrypatmine, and aeurginasin as measured by LC-MS from three different samples. The samples are from transgenic PsiD fungi (sample Nos. 1771 and 1772) and wild type fungi (sample No. 1773).

Example 6. Generating PsiH2 transgenic fungus for the production of alkaloids

[505] Fungal material is taken from a Psilocybe cubensis fungus. The material is prepared for transformation with a PsiH2 plasmid as described in Example 2. The PsiH2 plasmid contains a PsiH2 gene from P. cyanescence having SEQ ID NO. 15, or a PsiH2 gene from P. tampanensis having SEQ ID NO: 16. The PsiH2 plasmid is incorporated into the fungal cell by transformation as described in Example 2. The PsiH2 gene is driven by a GDP promoter having SEQ ID NO:

30. An intron is disposed between the PsiH2 gene and the promoter. The intron has SEQ ID NO:

31. Transformed colonies are transferred to fresh selection media every 15 days to selectively grow cells with the exogenous PsiH2 nucleic acid integrated. To confirm integration of the PsiH2 exogenous nucleic acid, a GUS assay and PCR analysis are performed.

[506] The mycelia are grown into mycelial masses from which primordia are produced. The alkaloidal content of the PsiH2 transgenic fungi can be evaluated by liquid chromatography/mass spectrometry to determine the alkaloids, and amounts of the alkaloids, that are present in the transgenic fungi. Briefly, fruiting bodies of PsiH2 transgenic fungi and non-transgenic fungi are dissected and transferred to 50 mL falcon tubes and snap frozen in liquid nitrogen. Wet weight is measured, and samples are maintained at -80 degrees Celsius. Samples are desiccated in a freeze drier at -45 degrees Celsius at 0.05 bar for 24 hours. The freeze-dried samples are then ground to a fine powder at room temperature using a mortar and pestle. Ground samples are transferred to a 50 mL tube and dry weight was measured. Approximately 7.1 grams of dry ground sample is transferred to a subsequent set of 50 mL tubes and sent for LC-MS analysis.

Example 7. Engineering transcriptional landscape to generate new compounds

[507] Transcriptional regulation is a genetic highway to acquire production capabilities for novel secondary metabolites or alter the existing biosynthesis pathways.

[508] FIG. 20 illustrates genes from the psilocybin cluster of six psilocybin-producing fungal species. The genes are coded by a gray gradient according to the annotation key provided. As illustrated, the gene order of the psilocybin cluster show discrepancies between the six psilocybin-producing species, which may be a result of genetic events such as inversions, duplications, etc., which suggests active re-arrangements and occurrences of horizontal gene transfer within in the cluster. These discrepancies suggest alternative routes of psilocybin regulation and production. All psilocybin-producing fungi contain a transcriptional regulator called PsiR within the known psilocybin cluster or elsewhere in the genome. As illustrated, Psilocybe cubensis may be genetically modified to incorporate an exogenous nucleic acid encoding one or more additional copies of PsiR 2003.

[509] PsiR is a basic helix loop helix (bHLH) transcriptional regulator expressed in mycelium and fruiting bodies of fungi. The expression of PsiR coincides with psilocibin production. bHLH binds to DNA at a consensus hexanucleotide sequence known as an E-box CANNTG, where N is any nucleotide (SEQ ID NO: 150). Binding of the PsiR regulator to the E-Box of a gene element results in an upregulation of gene expression.

[510] Of the psilocybin cluster genes, four (4) genes, PsiD, PsiH, PsiM, PsiT2, contain at least one E-box motif in their promoters (PsiTl has two), whereas PsiP contains 4 E-box motifs (500bp upstream of ATG). PsiL and PsiK do not contain the E-box motif in their upstream regions. A full transcript of PsiR is present in fruiting bodies but not in mycelium suggesting that PsiR is differentially regulated during the fungi life cycle. Induced expression of PsiR in mycelium can be used to upregulate alkaloid production at an earlier stage of the fungal life cycle. [5H] A fungal cell is genetically modified by introducing an exogenous nucleic acid into the fungal cell, wherein the exogenous nucleic acid encodes PsiR. The exogenous nucleic acid includes a GDP promoter driving a PsiR gene (SEQ ID NOS: 7 or 14). The GDP promoter has SEQ ID NO: 30. An intron is disposed between the GDP promoter and the PsiR gene. The intron has SEQ ID NO: 31. The ectopic overexpression of PsiR upregulates the psilocybin biosynthesis pathway and also activates/upregulates other biosynthesis pathways including novel components and entourage alkaloids via targeted binding to E-box motifs.

[512] Example 8. Genetically engineered reduction of psilocybin degradation products

[513] To increase production of a bioactive compound, e.g., psilocybin, the bioactive compound is produced simultaneously with an inhibitor of its own degradation, thereby increasing the overall production of the bioactive compound. An enzyme that produces a P- carboline core is overexpressed in Psilocybe cubensis to enhance production of bioactive compounds.

[514] P-carbolines are neuroactive compounds that inhibit monoamine oxidases which degrade psilocybin in human body. They are present in P. cubensis (i.e., harmala alkaloids such as harmane and harmine) but at very low amounts (around 0.2 pg/g). They are part of entourage in Psilocybe to prevent psilocybin degradation in human body.

[515] FIGS. 21A and 21B shows P-carbolines biosynthesis pathways. P-carboline core construction requires a Pictet-Spengler cyclization process. FIG. 21A shows a pathway from bacteria, which is used to produce a P-carboline scaffold from L-tryptophan. FIG. 21B shows a related pathway from a plant, which involves condensation of tryptamine and secologanin to produce a tetrahydro-P-carboline compound.

[516] The P-carbolines biosynthesis pathway diverged from the same building block (Trp) as psilocybin but produces dissimilar compounds; yet contribute to the same pharmacological effects. Harmala alkaloids are also found in the Banisteriopsis caapi vine, the key plant ingredient in the sacramental beverage Ayahuasca.

[517] An enzyme that produces a P-carboline core is overexpressed in Psilocybe cubensis to enhance production of bioactive compounds. The enzyme is encoded by a bacterial gene, McbB gene, which has SEQ ID NO: 126. To induce expression of the McbB gene ins a fungal cell, the gene is driven by a GDP promoter with SEQ ID NO: 31. The induced expression of the McbB gene the fungal cell leads to production of a useful alkaloid, DMT (N, N-Dimethyltryptamine), which when delivered to patients suffering from mental health disorder results in reduced symptoms. [518] Additional transgenic fungi are genetically modified to induce expression of a plant enzyme that produces P-carboline in Psilocybe cubensis. This can result in the enhanced production of DMT, which is found in some plant species. The enzyme is encoded by the plant gene, strictosidine synthase (STST) from Catharanthus roseus, which has SEQ ID NO: 125. Induced expression of the STST gene leads to production of DMT in the fungal cell.

Example 9. Engineering DMTP in Psilocybe.

[519] Psilocybe cubensis is genetically modified to produce DMT. DMT is found in several plants and is one of the active ingredients in Ayahuasca. DMTP (N,N,dimethyl-L-tryptophan) can be decarboxylated metabolically into DMT after ingestion through the action of aromatic L- amino acid decarboxylase (AAAD). This disclosure includes the discovery that PsTrpM, and in particular, PsTrpM from Psilocybe serbica. This can be used to produce DMT in a genetically modified Psilocybe cubensis.

[520] FIG. 22 illustrates the methyl transfer steps of TrpM and PsiM during biosynthetic pathways to N,N-dimethyl-L-tryptophan and psilocybin, respectively. TrpM originates from a retained ancient duplication event of a portion of the egtDB gene (latter required for ergothioneine biosynthesis and processively trimethylates L-histidine), and is phylogenetically unrelated to PsiM.

[521] The TrpM gene from the fungus Psilocybe serbica is introduced into Psilocybe cubensis on an exogenous nucleic acid. The gene comprises SEQ ID NO: 124, which is driven by a GDP promoter, SEQ ID NO: 30. By expressing the TrpM from the exogenous nucleic acid inside Psilocybe cubensis, DMT is produced. The genetically modified Psilocybe cubensis fungus can further be modified to, e.g., produce increased amounts of L-tryptophan (e.g., introducing an exogenous nucleic acid encoding PsiD), or downregulate enzymatic pathways that use L- tryptophan, to thereby produce greater amounts of DMT.

Example 10. PsiD-independent psilocybin biosynthesis pathway to produce DMT in Psilocybe

[522] A codon optimized Indolethylamine N-methyltransferase from homo sapiens HsINMT (SEQ ID NO: 129) and a codon optimized aromatic L-amino acid decarboxylase (AAAD) from P. cubensis (SEQ ID NO: 122) are introduced into Psilocybe cubensis. The resulting fungi is crossed with a fungi tryptophan decarboxylase as described above and may be further crossed with a line producing more P-carbolines. AAAD is a noncanonical calcium-activatable aromatic amino acid decarboxylase. AAADs are responsible for alkylamine production in kingdoms of life other than fungi, like L-DOPA decarboxylase which catalyzes the first step in the biosynthesis of monoamine neurotransmitters. AAAD in P. Cubensis shows substrate permissiveness towards L-phenylalanine, L-tyrosine, and L-tryptophan. In Psilocybe mushrooms, L-tryptophan decarboxylation is catalyzed by a neofunctionalized phosphatidylserine decarboxylase-like enzyme (PsiD) rather than by AAAD. Here, however, PcAAAD is used to mediate de novo psilocybin biosynthesis under the control of endogenous calcium signaling and/or elevated environmental calcium concentration. HsINMT (HsINMT, 262 aa) di-methylates tryptamine into DMT.

Example 11. Exploiting gene diversity of PsiM to generate alkaloids

[523] A phylogenetic analysis of PsiM was performed. In particular, amino acid sequences of PsiM gene products of four species of psilocybin-producing fungi: Psilocybe cubensis, Psilocybe azurescens, Psilocybe cyanescens, and Psilocybe tampanensis were aligned and compared.

[524] FIG. 23 shows a comparison of PsiM gene products from four different psilocybin- producing fungi. The comparison of the aligned sequences reveals diversity in the gene products of PsiM among the different fungal species.

[525] PsiM catalyzes iterative methyl transfer to the amino group of norbaeocystin to yield psilocybin via a monomethylated intermediate, baeocystin. Psilocybe azurescence is amongst the most potent psilocybin-producing mushrooms. PsiM is regulated on transcriptional level and more copies of PsiM lead to its over expression.

[526] Accordingly, to screen for production of higher amounts of alkaloids, one or more copies of the PsiM gene from P. azurescence (SEQ ID NO: 121) is integrated into a nucleic acid and introduced into the genome of Psilocybe cubensis. The overexpression of the heterologous PsiM gene is screened for production of increased amounts of alkaloids. The overexpression of the heterologous PsiM leads to enhanced production of psilocybin.

Example 12. Genetically engineered reduction of psilocybin degradation products

[527] To increase production of a bioactive compound, e.g., psilocybin, the bioactive compound is produced simultaneously with an inhibitor of its own degradation, thereby increasing the overall production of the bioactive compound. An enzyme that produces a P- carboline core is overexpressed in Psilocybe cubensis to enhance production of bioactive compounds.

[528] P-carbolines are neuroactive compounds that inhibit monoamine oxidases which degrade psilocybin in human body. They are present in P. cubensis (i.e., harmala alkaloids such as harmane and harmine) but at very low amounts (around 0.2 pg/g). They are part of entourage in Psilocybe to prevent psilocybin degradation in human body.

[529] FIGS. 21A and 21B shows P-carbolines biosynthesis pathways. P-carboline core construction requires a Pictet-Spengler cyclization process. FIG. 21A shows a pathway from bacteria, which is used to produce a P-carboline scaffold from L-tryptophan. FIG. 21B shows a related pathway from a plant, which involves condensation of tryptamine and secologanin to produce a tetrahydro-P-carboline compound.

[530] The P-carbolines biosynthesis pathway diverged from the same building block (Trp) as psilocybin but produces dissimilar compounds; yet contribute to the same pharmacological effects. Harmala alkaloids are also found in the Banisteriopsis caapi vine, the key plant ingredient in the sacramental beverage Ayahuasca.

[531] An enzyme that produces a P-carboline core is overexpressed in Psilocybe cubensis to enhance production of bioactive compounds. The enzyme is encoded by a bacterial gene, McbB gene, which has SEQ ID NO: 126. To induce expression of the McbB gene ins a fungal cell, the gene is driven by a GDP promoter with SEQ ID NO: 31. The induced expression of the McbB gene the fungal cell leads to production of a useful alkaloid, DMT (N, N-Dimethyltryptamine), which when delivered to patients suffering from mental health disorder results in reduced symptoms.

[532] Additional transgenic fungi are genetically modified to induce expression of a plant enzyme that produces P-carboline in Psilocybe cubensis. This can result in the enhanced production of DMT, which is found in some plant species. The enzyme is encoded by the plant gene, strictosidine synthase (STST) from Catharanthus roseus, which has SEQ ID NO: 125. Induced expression of the STST gene leads to production of DMT in the fungal cell.

Example 13. Engineering DMTP in Psilocybe

[533] Psilocybe cubensis is genetically modified to produce DMT. DMT is found in several plants and is one of the active ingredients in Ayahuasca. DMTP (N,N,dimethyl-L-tryptophan) can be decarboxylated metabolically into DMT after ingestion through the action of aromatic L- amino acid decarboxylase (AAAD). This disclosure includes the discovery that PsTrpM, and in particular, PsTrpM from Psilocybe serbica. This can be used to produce DMT in a genetically modified Psilocybe cubensis.

[534] FIG. 22 illustrates the methyl transfer steps of TrpM and PsiM during biosynthetic pathways to N,N-dimethyl-L-tryptophan and psilocybin, respectively. TrpM originates from a retained ancient duplication event of a portion of the egtDB gene (latter required for ergothioneine biosynthesis and processively trimethylates L-histidine), and is phylogenetically unrelated to PsiM.

[535] The TrpM gene from the fungus Psilocybe serbica is introduced into Psilocybe cubensis on an exogenous nucleic acid. The gene comprises SEQ ID NO: 124, which is driven by a GDP promoter, SEQ ID NO: 30. By expressing the TrpM from the exogenous nucleic acid inside Psilocybe cubensis, DMT is produced. The genetically modified Psilocybe cubensis fungus can further be modified to, e.g., produce increased amounts of L-tryptophan (e.g., introducing an exogenous nucleic acid encoding PsiD), or downregulate enzymatic pathways that use L- tryptophan, to thereby produce greater amounts of DMT.

Example 14. Metabolite Analysis of engineered fungal cells

[536] Triplicate samples of the engineered fungal cells were extracted with methanol and submitted for High-Resolution Accurate Mass Spectrometry (HRAMS). This analysis was conducted using XXX. Six different batches of a genetically modified mushroom over expressing PsiD were cultivated and then tested for alkaloid abundance measured in mg/gram of mushroom sample. Measurements were performed in triplicate, referred to herein as “consistency tests” (CT) and each alkaloid abundance was compared between mushroom batches.

[537] One set of samples were grown under “shaken” conditions, alkaloid content was measured according to the above protocol.

Example 15. Quantitative Metabolite Analysis of engineered fungal cells

[538] Triplicate samples of the engineered fungal cells were extracted with methanol and submitted as powder samples to determine the alkaloid concentration by liquid Chromatography Mass Spectrometry (LCMS). The experimental samples were analyzed against analytical standards purchased commercially from Cayman Chemicals. Calibration standards, quality control (QC) solutions and blank matrix solutions with spiked analytical standards were prepared using conventional methods. Solution preparations indicating scale and concentrations are described below in TABLE 30-TABLE 32, and results shown in FIGs. 28A-28F.

TABLE 30. Alkaloid Concentrations in Genetically Modified Fungi

TABLE 31. Average Alkaloid Concentrations in Genetically Modified Fungi

TABLE 32. Dilution Quantifications of Alkaloid Concentrations in Genetically Modified Fungi

Assuming a 100% Recovery in ppm (ug/g)

ND: Not detected even at a dilution of 1 : 10.

Example 16. Sample Preparations for Quantitative Metabolite Analysis of engineered fungal cells

Analytical Standards

[539] The experimental samples were analyzed against analytical standards purchased commercially from Cayman Chemicals. Stock solutions of the analytical standards of each alkaloid were prepared by diluting the solid material in anhydrous methanol (MeOH) to a concentration of 1.0 mg/mL. In the case of norbaeocystin analysis, the solid fungal material samples were diluted in a 1 : 1 solution of anhydrous dimethylsulfoxide (DMSO): anhydrous MeOH at a concentration of 0.5 mg/mL. Internal standard solutions were purchased as lOOpg/mL solution or prepared in water by dissolving 1 mg of each internal standard into lOmL of water. The internal standards included psilocin-Dio and psilocybin-D4, acquired from Merck and Cambridge Bioscience respectively. Internal standard spiking solutions were prepared as 1 mL of a 2 pg/mL solution of Psilocin-Dio and Psilocybin-D4 in water from stock solutions (100 pg/mL). The internal MeOH extraction solvent was prepared as a 4 pg/mL of Psilocin-DlO and Psilocybin-D4 in methanol from the IS stock solutions 100 pg/mL. 10 mL of this extraction solvent is required for each 100 mg mushroom sample and 500 pL for each 5 mg sample.

Blank Matrix Extraction Samples

[540] Cryomilled generic mushrooms (100 mg of each mushroom sample) were diluted with 10 mL anhydrous MeOH. The sample solutions underwent shaking for 10 minutes at 700 rpm, centrifuged at 4000 rpm for 4 minutes, filtered on 0.22 pm PTFE filters, and stored at -80 degrees Celsius. Calibration of quality control (QC) samples were then prepared by diluting the stored sample solutions in water (1 : 100).

Fungal Samples

[541] Each experimental fungal sample was stored at -80 degrees prior to extraction. Once removed from the freezer, all samples were extracted immediately.

Sample extraction 100 mg Scale

[542] Mushroom extraction was carried out on a 100 mg scale. 10 mL of methanol extraction solvent was added to the powdered mushroom sample. The samples were covered in foil, underwent shaking for 10 minutes at 700 rpm, centrifuged at 4000 rpm for 4 minutes, a 1 mL aliquot of each sample was filtered on 0.22 pm PTFE filters, the filters were washed 3 times with 200 pL of MeOH and combined together in one vial. The solvent was evaporated under nitrogen gas at room temperature. The samples were reconstituted with 1 mL of a phosphate buffer (pH 7- 7.3) and then diluted lOOx in water prior to analysis by LCMS/MS.

Sample extraction - 5 mg Scale

[543] Mushroom extraction was carried out on a 5 mg scale. 500 pL of methanol extraction solvent was added to the powdered mushroom sample. The samples were covered in foil, underwent shaking for 10 minutes at 700 rpm, centrifuged at 4000 rpm for 4 minutes, a 200 pL aliquot of each sample was filtered on 0.22 pm PTFE filters, the filters were washed 3 times with 200 pL of MeOH and combined together in one vial. The solvent was evaporated under nitrogen gas at room temperature. The samples were reconstituted with 200 pL of a phosphate buffer (pH 7-7.3) and then diluted lOOx in water prior to analysis by LCMS/MS.

Liquid chromatographic conditions

[544] The LC system used was a Waters Acquity UPLC with binary pump. A phenyl-hexyl 2 x 100 mm, 3 pm column at 60 °C was used to separate the alkaloids. The aqueous mobile phase was 10 mM ammonium acetate, pH adjusted to 4 with acetic acid. The organic mobile phase was methanol. The gradient started at 1% organic mobile phase held for 0.2 minutes, going to 99% organic in 1.8 minutes, held for 0.5 minutes, then returns to 1% in 0.1 minutes and then held for 0.4 minutes. Total runtime was 3 minutes. The flow rate was 0.750 mL/min and 1 pL sample was injected.

Liquid chromatographic conditions

[545] Positive electrospray ionization was used on a Waters TQ-XS mass spectrometer. The MRM transitions monitored were as described in TABLE 34:

TABLE 33. MRM transitions and MS abundancies for analytical alkaloid analysis

MS methods are subject to change dependent on instrumentation

Example 17. RNP CRISPR transformation with Mycelium

[546] Fungal organisms are genetically modified using CRISPR systems to produce new or rare alkaloids. Fungal material from the fungal organisms is prepared for genetic modification with a CRISPR system. The CRISPR system is provided into the fungal material in the form of active ribonucleoprotein (RNP) consisting of Cas endonuclease pre-complexed with guide RNA or one or more nucleic acids encoding the Cas endonuclease and guide RNA.

[547] Fungal protoplasts are extracted from mycelial mass of Psilocybe cubensis. In particular, the Psilocybe cubensis mycelia are maintained on potato dextrose agar (PDA) at 25 degrees Celsius in the dark. The mycelia (cells 3 weeks old or less) are cut into small blocks from agar plates and added to 100 mL potato dextrose broth (PDB) media. The mycelium cultures are incubated in a shaker incubator (at 175 rpm) for six (6) days in the dark at 28 degrees Celsius. [548] Following incubation, six (6) day old Psilocybe cubensis are transferred to fresh PDB medium and homogenized using an Ultra-Turrax homogenizer 24 hours before inoculation. Hyphal fragments are transferred to fresh PDB and grown for 24 hours to give a uniform mycelial slurry under same conditions as the originally maintained mycelia sample.

[549] RNPs are provided in range of about 60-100 nM to transform into fungal protoplasts. About 100,000 - 200,000 fungal protoplasts are used for transformation. The transformation is made with approximate 20 nM of donor DNA (exogenous nucleic acid) encoding a psilocybin synthesis gene, PsiD (SEQ ID NO: 90), which is driven by a GDP promoter (SEQ ID NO: 406). To ensure that the RNPs cross the fungal cell membrane during protoplast transformation, a surfactant Triton X-100 is used. The surfactant, Triton X-100, is used to improve cell membrane permeability. Surprisingly, the gene editing systems disclosed herein still function in the presence of Triton X-100.

[550] Protoplast solutions with 60-100 nM of RNPs can be mixed with Triton X-100 (0.006% (w/v) final concentration in transformation reaction, and the incubation time (at 20 degrees Celsius) is prolonged to 25 min before the mixture is transferred to the selective medium. The efficiency of RNP transformation into Psilocybe cubensis can reached over 25 cell colony forming units per 100,000 - 200,000 protoplasts with 60-100 nM RNPs.

Example 18. Methods using Agrobacterium to deliver CRISPR systems

[551] Agrobacterium tumefaciens can be used to deliver CRISPR systems into target fungal cells as described below.

[552] A. tumefaciens strains LBA4404/AGL1 carrying a CRISPR plasmids are grown for 24- 48 hours in Lysogeny broth (LB) medium supplemented with appropriate antibiotics prior to inoculation. The CRISPR plasmids include a Cas9 endonuclease having SEQ ID NO: 96 with a nuclear localization signal comprising SEQ ID NO: 97, and one or more guide polynucleotides. On the day of inoculation, bacterial cultures were diluted to an optical density of 0.15 at 660 nm with agrobacterium induction medium (AIM) (Induction medium (AIM) [MM containing 0.5% (w/v) glycerol, 0.2 mM acetosyringone (AS), 40 mM 2-N-morpholino-ethane sulfonic acid (MES), pH 5.3) and grown for an additional 5-6 hours in a shaker incubator at 28 degrees Celsius.

[553] For transformation A 25 mL aliquot of the mycelial suspension (uniform mycelial slurry) is mixed with 25 mL of a bacterial cultures the CRISPR plasmids. 1 mL of each of the mixtures is spread on cellophane discs following vacuum treatment and overlaid on agar plates and incubated at 25 degrees Celsius for 72-92 hours in the dark. Agrobacterium contamination level was observed after 72 hours. Co-cultivation was stopped if agrobacterium was visible, otherwise, incubation was continued up to 92 hours.

[554] After cocultivation, cellophane discs are transferred to Selection 1, which included PDA medium containing 200 mg/L Timentin to kill residual agrobacterium cells and 80 mg/L Hygromycin to select fungal transformants. Selection 1 was incubated for 10-15 days at 25 °C. After Selection 1, individual colonies were transferred to PDA medium containing 80 mg/L Hygromycin and 200 mg/L Timentin (Selection 2).

Example 19. Confirmation of transgenic fungi produced by CRISPRs

[555] Transformed colonies generated according to Examples 1 and 2 demonstrate significant growth and are transferred to new selection media every 15 days to selectively grow cells with the exogenous nucleic acid integrated. To confirm integration of the exogenous nucleic acid encoding tryptophan decarboxylase, a GUS assay, short for “beta-glucuronidase”, and PCR analysis are performed. Both assays confirm transformation of the exogenous nucleic acid.

[556] Transgenic and non-transgenic colonies are cultured on selection plates with Hygromycin (80 mL) and Timentin (200 mg/L).

[557] The mycelia are grown into mycelial masses from which primordia are produced. Each mass is wrapped in aluminum foil and placed in the dark at 27 degrees Celsius for incubation. After 18 days, the cakes are transferred into bigger pots with vents open. For one set of the cakes, the pots are kept in the cup with wet casing, and the second set was removed from their cups with the casing on top.

[558] Wild-type mycelia and transgenic mycelia that express elevated levels of PsiD exhibit a phenotype that is visually district from the comparable wild-type mycelia of the non-transgenic wild-type mycelia. The phenotype comprises a blue coloration that is district from the wild-type mycelia and reflects light having a wavelength of between about 450 nanometers and 500 nanometers. The phenotype is measurable using a spectrophotometer. The spectral reflectance, of the genetically modified organism, in the wavelength region from 400 nm to 525 nm (the blue regions) is high, and the spectral reflectance for wavelengths longer than 550 nm is low. Conversely, the wild-type mycelia has a spectral reflectance in the wavelength region from 400 nm to 525 nm, which is substantially lower than the transgenic mycelia.

Example 20. Additional Alkaloid analyses of transgenic fungi generated by CRISPRs

[559] The alkaloid content of the genetically modified mushrooms is analyzed by liquid chromatography/mass spectrometry to determine amounts of alkaloids present in the transgenic fungi. Liquid chromatography-mass spectrometry (LC-MS) is an analytical method that combines the features of liquid-chromatography and mass spectrometry to identify different substances within a test sample. To accurately assess the alkaloids, present in transgenic fungi, LC-MS analyses are conducted at two independent facilities.

[560] Genetically engineered and control P. cubensis fungi are cultivated in a laboratory to obtain fruiting bodies. The cultured fungi are cut at the base of the stipe and freeze dried overnight before homogeneously powdered using a mortar and pestle. Both the cap and stipe are analysed together for alkaloid content. Quantitative testing of psilocybin, psilocin, baeocystin, norbaeocystin and tryptophan, and semi-quantitative testing for Aeruginascin and Norpsilocin are performed using high performance liquid chromatography - tandem mass spectrometry (HPLC-MS/MS). For quantitative analysis, alkaloid content is compared to known concentrations of psilocybin, psilocin, baeocystin, norbaeocystin and tryptophan synthetic chemical standards.

[561] Alkaloids are measured in PsiD transgenic and wild-type fungi by LC-MS. Relative amounts of alkaloids are shown in FIGs. 18A-18B, and FIGs 19A-19D. Norbaeocystin is 34.7- 53.7 times upregulated as compared with a comparable wild-type fungus, baeocystin is 17.5-12.5 times upregulated as compared with a comparable wild-type fungus, norpsilocin is 10-12.1 times upregulated as compared with a comparable wild-type fungus, psilocybin is 2.2-2.3 times upregulated as compared with a comparable wild-type fungus, and psilocin is 5.3-5.4 times upregulated in the PsiD transgenic fungi as compared to a comparable wild-type fungus as compared with a comparable wild-type fungus.

Example 21. Protoplast Extraction

Protoplasts were prepared for transfection according to the following protocol. On day 1, small blocks of mycelium were inoculated into a 100 mL liquid potato dextrose broth (PDB) medium. This method was consistent with general purpose growth of fungal cells. On Day 3 or Day 4, the mycelial blocks were blended using low to medium speed in order to homogenize the contents of the sample in a falcon tube. The resulting homogenized mycelia samples in solution were then diluted to 150 mL, grown at 28 degrees Celsius at 150 rpm for 16-18 hours. On Days 4 or 5, the homogenized mycelia samples were transferred to new falcon tubes and underwent spinning at 1800 rpm for 5 minutes. The supernatant was disposed. The homogenized mycelia samples were resuspended in an enzyme solution comprising Yatalase with VinoTase or Yatalase with a Protoplast. The resulting suspension was subsequently incubated at 30 degrees Celsius at shaking conditions of 55 rpm for approximately 10-16 hours. On Days 7 or 8, protoplast were separated from the intact mycelium and cell wall debris by filtering the protoplast suspension through four to six layers of sterile cheese cloth followed by filtering the protoplast suspension through sterile nylon fabric (40 pm cell strainer). The filtrate was centrifuged at 2000 rpm for 10 minutes at 4 degrees Celsius. The supernatant was subsequently collected. The pellets in the remaining solution were shaken, gently. MM buffer (10 mL) were added to each pellet while on ice. Protoplasts were then counted, and density adjusted to 10⁷/mL with cold MMC and kept on ice.

Example 22. Protoplast Transfection for RNP replacement (MMEJ)

Protoplast transfections were carried out for single complex, double complex for protoplast transfection with Plasmid DNA. All transfections included approximately 0.5-1.0 x 10⁶ protoplasts for each transformation. All steps are conducted on ice and in darkness unless otherwise indicated. Control samples were run in parallel comprising no guide RNAs or DNA. Single Complex

[562] An RNP complex is prepared using a Cas9: guide RNA ratio of 1 :3. In a solution, an RNP complex buffer, a Cas9_NLS_GFP, a first guide RNA, a second guide RNA, and water are added together. The mixture is then pre-incubated with thermocycler at 35 degrees Celsius for 3 minutes, and then 23 degrees Celsius for 12 minutes. Without cooling, the DNA template is added and gently mixed. The protoplast suspension is separately prepared and kept on ice until the RNP is ready. The RNP complex and DNA repair template are added into the cold protoplast suspension and mixed gently. The resulting mixture is placed on ice in the dark for approximately 10 minutes. A sterilized and filtered PEG solution is added in a 1 : 1 ratio to the resulting mixture. The resulting mixture is incubated on ice for approximately 20 minutes. An additional aliquot of a sterilized and filtered PEG solution is added in a 1 : 1 ratio and subsequently incubated on ice for approximately 20 minutes. The solution is left to warm to room temperature, additional PEG solution at room temperature is added and the mixture is incubated at 30 degrees Celsius for 10 minutes, and then at room temperature for 20 minutes in the dark. The reaction is stopped by adding STC and incubating at 26 degrees Celsius to recover and regenerate back cell walls overnight. The incubated mixture is then aliquoted 50 pl and placed directly onto PDAS + Hygromycin (50 -80mg/L) + Timentin (160mg/L). After approximately 10 days, the colonies with the hygromycin resistance gene will begin to grow on the hygromycin plates.

Double Complex

[563] The above protocol is used with a Cas9: guide RNAs ratio of 1 :3 for every guide used (e.g., for two guides it will be 2:3:3 for Cas9: first guide RNA and second guide RNA). Protoplast transfection with Plasmid DNA

[564] A protoplast suspension is kept on ice. Plasmid DNA is added into the cold protoplast and mixed gently for 2 minutes. The mixture is placed on ice in the dark for approximately 10 minutes. A cold sterilized and filtered PEG solution is added and placed on ice in the dark for 30 minutes and then moved to room temperature for 10 minutes. An additional aliquot of a sterilized and filtered PEG solution (left to warm to room temperature for 10 minutes) is added and incubated for 20-30 minutes in the dark. STC buffer is then added at room temperature and the resulting mixture is incubated at 26 degrees Celsius for 1 hour. Different dilutions of the mixture with additional STC buffer are plated and incubated. Undiluted mixture is left to incubate overnight at 26 degrees Celsius. The reaction is then analyzed for GFP and mCherry positive transfected protoplast. An overlay of PDAS+ Hygromycin (100 mg/L) + Timentin (160mg/L) added and incubated at 28 degrees Celsius for 2 weeks.

[565] As one of skill in the art will readily appreciate. This disclosure has been presented for purposes of illustration and description. The discussion above is not intended to limit the disclosure to the form or forms disclosed herein. Although the description of the disclosure has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the present disclosure.

INCORPORATION BY REFERENCE

[566] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Absent any indication otherwise, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. An engineered fungal cell comprising an increased expression of at least one gene product encoded by a gene selected from the group consisting of: PsiL, TrpE, PsiH2, and a combination of any of these.

2. The engineered fungal cell of claim 1, wherein the gene comprises a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 15, 16, 127, 128, and 322.

3. The engineered fungal cell of claim 1, wherein the genes each independently comprise a sequence that has 100% identity to any one of SEQ ID NO: 15, 16, 127, 128, and 322.

4. The engineered fungal cell of claim 1, that is comprised in a multicellular organism.

5. The multicellular organism of claim 4, that is from division Basidiomycota.

6. The multicellular organism of claim 5, that is a species selected from the group consisting of: Psilocybe cyanescens, Psilocybe azurescence, and Psilocybe tampanensis.

7. The multicellular organism of claim 4, that is the species Psilocybe cubensis.

8. The engineered fungal cell of claim 1, that comprises an alkaloid.

9. The engineered fungal cell of claim 8, wherein the alkaloid is selected from the group consisting of: N,N-dimethyltryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-/,- tryptophan, 5 -hydroxy -/.-tryptophan, 7-hydroxy-Z-tryptophan, isonorbaeocystin, 4- phosporyloxy-N,N-dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol- 3-yl)-N,N,N-trimethylethan-l-aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, harmine, harmane, a P-carboline, and a salt of any of these.

10. The engineered fungal cell of claim 1, which has an increased expression of an alkaloid as compared to a comparable fungal cell without the genes.

11. A composition comprising: a genetically modified organism comprising a heterologous PsiH2 gene that encodes a tryptamine monooxygenase, wherein the tryptamine monooxygenase is expressed in an amount sufficient to produce an alkaloid or a precursor thereof.

12. The composition of 11, wherein the heterologous PsiH2 gene comprises a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOS: 15 or 16.

13. The composition of claim 11, wherein the heterologous PsiH2 gene comprises a sequence that has 100% identity to SEQ ID NO: 15 or 16.

354

14. The composition of claim 11, wherein the genetically modified organism is a multicellular organism.

15. The composition of claim 14, wherein the multicellular organism comprises a fungus.

16. The composition of claim 15, wherein the fungus is from division Basidiomycota.

17. The composition of claim 11, wherein the tryptamine monooxygenase comprises a tryptamine 4-monooxygenase from one of Psilocybe cyanescens, Psilocybe azurescence, or Psilocybe tampanensis.

18. The composition of claim 15, wherein the fungus is from Psilocybe cubensis and wherein the tryptamine monooxygenase is not naturally expressed at a detectable amount in said fungus.

19. The composition of claim 8, wherein the alkaloid is not expressed in a detectable amount in a comparable organism without the genetic modification.

20. The composition of claim 19, wherein the alkaloid is a compound selected from N,N- dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-Z-tryptophan, 5-hydroxy-/.- tryptophan, 7-hydroxy-Z-tryptophan, isonorbaeocystin, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan- 1 -aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, harmine, harmane, P- carboline, and a salt thereof.

21. The composition of claim 20, wherein the genetic modification results in increased expression of the alkaloid as compared to a comparable organism without the genetic modification.

22. The composition of claim 21, wherein the alkaloid is a compound according to Formula

a tautomer thereof, or a pharmaceutically acceptable salts of any of the foregoing wherein: -R¹ is H or -CH₃;

355 -R² is H, -OH, -OP(O)(OM)₂, -C(O)CH₃, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

-R³ is H, -OH, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

-R⁴ and R⁵ are each independently H, -OH, or -OCH₃;

-R^A is H, -COOH;

-R^x, R^y, and R^z are each independently H, -CH₃, -C(O)CH₃, -CH₂CH₃, or -CH(CH₃)₂; and -M is H, Li⁺, Na⁺, K⁺ or is absent. The composition of claim 22, wherein the alkaloid is a compound selected from psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, isonorbaeocystin, and a salt thereof. The composition of claim 11, further comprising a modification that results in increased expression of a helix-loop-helix transcription factor that binds to an E-box motif as compared to a comparable organism without said modification. The composition of claim 24, wherein the helix-loop-helix transcription factor is encoded by an exogenous gene that comprises a sequence that is at least 95% identical to one of SEQ ID NOS: 7 or 14. The composition of claim 24, wherein the modification results in upregulated expression of a gene product comprising any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, or a psilocybin-related phosphotransferase, as compared to a comparable organism without the modification. The composition of claim 26, wherein the gene product is encoded by any one of PsiD, PsiH, PsiM, PsiTl, PsiT2, PsiR, or PsiP. The composition of claim 1, further comprising a heterologous PsiR gene that encodes a helix-loop-helix transcription factor that, when expressed, interacts with a regulatory element of one or more genes, wherein the regulatory element comprises SEQ ID NO: 7 or 14. The composition of claim 28, wherein the one or more genes comprise any one of PsiD, PsiH, PsiM, PsiTl, PsiT2, or PsiP. The composition of claim 1, further comprising an exogenous nucleic acid that results in any one of increased tryptophan decarboxylation, increased tryptamine 4-hydroxylation, increased 4-hydroxytryptamine O-phosphorylation, or increased psilocybin production via sequential N-methylations, as compared to a comparable organism without the exogenous nucleic acid. The composition of claim 20, wherein the genetically modified organism comprises a fruiting body of a fungus. The composition of claim 20, wherein the genetically modified organism comprises a nonfruiting body of a fungus. The composition of any one of claims 20-32, wherein the genetic modification is accomplished by an agrobacterium-mediated insertion of an exogenous sequence into the genetically modified organism. The composition of any one of claims 20-32, wherein the genetic modification is accomplished by contacting a fungal cell with a gene editing system. The composition of claim 34, wherein the gene editing system comprises one of a CRISPR enzyme, TALE-Nuclease, transposon-based nuclease, Zinc finger nuclease, meganuclease, Mega-TAL or DNA guided nuclease. The composition of claim 34, wherein the gene editing system comprises a guide polynucleotide that targets one of SEQ ID NOS: 60-116. A method of treatment, wherein the method comprises administering the composition of any one of claims 20-26 to a subject diagnosed with a health condition. The method of claim 37, wherein the health condition is depression, anxiety, post-traumatic stress, addiction, psychological distress including cancer-related psychological distress, a neurodegenerative disease, an expression of symptoms in a neurodivergent individual. A pharmaceutical composition comprising the composition of any one of claims 11-38, and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition is formulated as a dosage form for topical, oral, inhalation, or intestinal delivery. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition comprises an effective amount of the genetically modified organism or an extract of the genetically modified organism for treating a health condition. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition is formulated such that an effective amount of the composition for treatment of the health condition can be delivered in a single dose format. A nutraceutical composition comprising the composition of any one of claims 20-36 or an extract thereof. A food supplement comprising the composition of any one of claims 20-36 or an extract thereof. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition is a neuro-modulating agent. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition is administered for symptom management in neurodivergent individuals. The pharmaceutical composition of claim 39, wherein the pharmaceutical composition is administered in effective amount for the treatment of a neurodegenerative disease. A method of treatment, wherein the method comprises administering the composition of any one of claims 45-47 to a subject diagnosed with a health condition. A composition comprising: a genetically modified organism comprising an exogenous nucleic acid, wherein the exogenous nucleic acid encodes a heterologous tryptamine monooxygenase that is not present in a comparable organism without said exogenous nucleic acid. The composition of claim 49, wherein the exogenous nucleic acid comprises a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOS: 15 or 16. The composition of claim 49, wherein the exogenous nucleic acid comprises a sequence that has 100% identity to SEQ ID NO: 15 or 16. The composition of claim 49, wherein the genetically modified organism is a multicellular organism. The composition of claim 52, wherein the multicellular organism comprises a fungus. The composition of claim 53, wherein the fungus is from division Basidiomycota. The composition of claim 53, wherein the tryptamine monooxygenase comprises a tryptamine 4-monooxygenase from one of Psilocybe cyanescens, Psilocybe azurescence, or Psilocybe tampanensis. The composition of claim 53, wherein the fungus is from Psilocybe cubensis and wherein the tryptamine monooxygenase is not naturally expressed at a detectable amount in said fungus. The composition of claim 49, wherein the exogenous nucleic acid, when expressed, results in the genetically modified organism producing an alkaloid that is not expressed in a detectable amount in a comparable organism without the genetic modification. The composition of claim 57, wherein the alkaloid is a compound selected from N,N- dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-Z-tryptophan, 5-hydroxy-/.- tryptophan, 7-hydroxy-Z-tryptophan, isonorbaeocystin, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan- 1 -aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, harmine, harmane, P- carboline, and a salt thereof.

59. The composition of claim 49, wherein the exogenous nucleic acid, when expressed, results in increased production of an alkaloid as compared to a comparable organism without the genetic modification.

60. The composition of claim 59, wherein the alkaloid is a compound according to Formula

-R¹ is H or -CH₃;

-R³ is H, -OH, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

-R⁴ and R⁵ are each independently H, -OH, or -OCH₃;

-R^A is H, -COOH;

61. The composition of claim 59, wherein the alkaloid is a compound selected from psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, isonorbaeocystin, and a salt thereof.

62. The composition of claim 49, wherein the genetically modified organism further comprises a modification that results in increased expression of a helix-loop-helix transcription factor that binds to an E-box motif as compared to a comparable organism without said modification.

63. The composition of claim 62, wherein the helix-loop-helix transcription factor is encoded by an exogenous gene that comprises a sequence that is at least 95% identical to one of SEQ ID NOS: 7 or 14. The composition of claim 62, wherein the modification results in upregulated expression of a gene product comprising any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, or a psilocybin-related phosphotransferase, as compared to a comparable organism without the modification. The composition of claim 64, wherein the gene product is encoded by any one of PsiD, PsiH, PsiM, PsiTl, PsiT2, PsiR, or PsiP. The composition of claim 49, wherein the genetically modified organism further comprises a heterologous PsiR gene that encodes a helix-loop-helix transcription factor that, when expressed, interacts with a regulatory element of one or more genes, wherein the regulatory element comprises an E-box motif. The composition of claim 53, wherein the one or more genes comprise any one of PsiD, PsiH, PsiM, PsiTl, PsiT2, or PsiP. The composition of claim 49, further comprising an exogenous nucleic acid that results in any one of increased tryptophan decarboxylation, increased tryptamine 4-hydroxylation, increased 4-hydroxytryptamine O-phosphorylation, or increased psilocybin production via sequential N-methylations, as compared to a comparable organism without the exogenous nucleic acid. The composition of claim 49, wherein the genetically modified organism comprises a fruiting body of a fungus. The composition of claim 49, wherein the genetically modified organism comprises a nonfruiting body of a fungus. The composition of any one of claims 49-70, wherein the exogenous nucleic acid is delivered into the genetically modified organism by an agrobacterium-mediated insertion of an exogenous sequence into the genetically modified organism. The composition of any one of claims 49-70, wherein the exogenous nucleic acid is integrated into the genome of the genetically modified organism by a gene-editing system, wherein the gene-editing system is delivered into the genetically modified organism by contacting said organism with a gene editing system. The composition of claim 72, wherein the gene editing system comprises one of a CRISPR enzyme, TALE-Nuclease, transposon-based nuclease, Zinc finger nuclease, meganuclease, Mega-TAL or DNA guided nuclease. The composition of claim 72, wherein the gene editing system comprises a guide polynucleotide that targets any one of SEQ ID NOS: 60-116. A method of treatment, wherein the method comprises administering the composition of any one of claims 49-61 to a subject diagnosed with a health condition. The method of claim 75, wherein the health condition is depression, anxiety, post-traumatic stress, addiction, or psychological distress including cancer-related psychological distress. A pharmaceutical composition comprising the composition of any one of claims 36-61, and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 77, wherein the pharmaceutical composition is formulated as a dosage form for topical, oral, inhalation, or intestinal delivery. The pharmaceutical composition of claim 77, wherein the pharmaceutical composition comprises an effective amount of the genetically modified organism or an extract of the genetically modified organism for treating a health condition. The pharmaceutical composition of claim 77, wherein the pharmaceutical composition is formulated such that an effective amount of the composition for treatment of the health condition can be delivered in a single dose format. A nutraceutical composition comprising the genetically modified organism of any one of claims 49-73, or an extract thereof. A food supplement comprising an extract of the genetically modified organism of any one of claims 49-61, or an extract thereof. A food supplement comprising an extract of the genetically modified organism of any one of claims 49-61, or an extract thereof. A composition comprising: a fungal cell comprising a genetic modification that results in the fungal cell expressing a heterologous tryptamine monooxygenase that is not expressed in a detectable amount in a comparable fungal cell without said genetic modification. The composition of claim 84, wherein the fungal cell is from division Basidiomycota. The composition of claim 84, wherein the heterologous tryptamine monooxygenase comprises a tryptamine 4-monooxygenase of a fungus from Psilocybe cyanescens, Psilocybe azurescence, or Psilocybe tampanensis. The composition of claim 84, wherein the fungal cell is from Psilocybe cubensis and wherein the heterologous tryptamine monooxygenase does not naturally occur at a detectable amount in the fungus. The composition of claim 84, wherein the heterologous tryptamine monooxygenase is encoded by a gene comprising a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOS: 15-16.

361 The composition of claim 84, wherein the heterologous tryptamine monooxygenase is expressed in the fungal cell at an amount sufficient to convert a compound into an alkaloid. The composition of claim 89, wherein the alkaloid is a compound selected from N,N- dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-Z-tryptophan, 5-hydroxy-Z- tryptophan, 7-hydroxy-Z-tryptophan, isonorbaeocystin, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan- 1 -aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, harmine, harmane, P- carboline and precursors, derivatives, and salts thereof. The composition of claim 84, wherein the expression of the heterologous tryptamine monooxygenase by the fungal cell results in increased production of the alkaloid as compared to a comparable fungal cell without the genetic modification. The composition of claim 91, wherein the alkaloid is a compound selected from psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, and a salt thereof. The composition of claim 84, wherein the fungal cell further comprises a modification that results in increased expression of a helix-loop-helix transcription factor that binds with an E-box motif as compared to a comparable organism without said modification. The composition of claim 93, wherein the helix-loop-helix transcription factor is encoded by a PsiR gene that comprises a sequence that is at least 95% identical to any one of SEQ ID NOS: 7 or 14. The composition of claim 93, wherein the modification results in upregulated expression of any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, or a psilocybin-related phosphotransferase, as compared to a comparable organism without said modification. The composition of claim 93, wherein the modification results in the upregulated expression of a gene product that is encoded by any one of PsiD, PsiH, PsiK, PsiM, PsiTl, PsiT2, PsiR, or PsiP, as compared to a comparable fungal cell without the modification. The composition of claim 93, wherein the tryptamine monooxygenase is encoded by an exogenous PsiH2 gene and is expressed in the fungal cell using an exogenous gene promoter, wherein the exogenous gene promoter is GPD. The composition of claim 93, wherein the tryptamine monooxygenase is encoded by a PsiH2 gene that comprises a sequence that is 100% identical to SEQ ID NO: 15 or 16. The composition of claim 93, wherein the fungal cell is from a fruiting body of a fungus.

362

. The composition of claim 93, wherein the fungal cell is from a non-fruiting body of a fungus. . The composition of claim 93, wherein the fungal cell further comprises a heterologous PsiR gene that encodes a helix-loop-helix transcription factor that, when expressed, interacts with a E-box motif of one or more genes encoding gene products involved in a biosynthesis pathway of an alkaloid. . The composition of claim 101, wherein the one or more genes are selected from PsiD, PsiH, PsiM, PsiTl, PsiT2, and PsiP. . The composition of claim 84, wherein the fungal cell further comprises an exogenous nucleic acid that results in any one of increased tryptophan decarboxylation, increased tryptamine 4-hydroxylation, increased 4-hydroxytryptamine O-phosphorylation, or increased psilocybin production via sequential N-methylations, as compared to a comparable fungal cell without the exogenous nucleic acid. . The composition of claim 84, wherein the fungal cell further comprises a modification that results in an increased expression of a gene product encoded by any one of PsiD, PsiM, PsiH, PsiK, or PsiR, as compared to a comparable fungal cell without the second modification. . The composition of any one of claims 84-104, wherein the genetic modification of the fungal cell is accomplished by an agrobacterium-mediated insertion of an exogenous sequence. . The composition of any one of claims 84-104, wherein the genetic modification is accomplished by contacting a fungal cell with a gene editing system. . The composition of claim 106, wherein the gene editing system comprises any one of a CRISPR enzyme, TALE-Nuclease, transposon-based nuclease, Zinc finger nuclease, meganuclease, Mega-TAL, DNA guided nuclease. . A pharmaceutical composition comprising the fungal cell or an extract of the fungal cell of the composition from any one of claims 84-107. . The pharmaceutical composition of claim 108, wherein the composition comprises an effective amount of the fungal cell or an extract of the fungal cell comprising an alkaloid for treating a health condition. . The pharmaceutical composition of claim 109, wherein the composition is formulated such that an effective amount of the composition for treatment of the health condition can be delivered in a single dose format.

363

1. A nutraceutical composition comprising an extract of the fungal cell or an extract of the fungal cell of any one of claims 84-94. . A food supplement comprising an extract of the fungal cell of any one of claims 84-94. 3. A method of treatment, wherein the method comprises administering the fungal cell or an extract of the fungal cell of any one of claims 84-94 to a subject diagnosed with a health condition. . The method of claim 113, wherein the health condition is depression, anxiety, post- traumatic stress, addiction, or psychological distress including cancer-related psychological distress. 5. A composition comprising: a genetically modified fungal cell comprising a genetic modification that comprises an exogenous nucleic acid encoding a helix-loop-helix transcription factor that binds to a regulatory element of one or more genes associated with an alkaloid biosynthesis pathway, wherein the exogenous nucleic acid comprises a sequence that has at least 95% identity to one of SEQ ID NOS: 7 or 14. . The composition of claim 115, wherein the regulatory element comprises a hexanucleotide sequence that is an E-box motif. . The composition of claim 115, wherein the one or more genes comprise PsiD, PsiH, PsiM, PsiTl, PsiT2, or PsiP. 8. The composition of claim 115, wherein the genetic modification results in upregulated expression of any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, or a psilocybin-related phosphotransferase, as compared to a comparable fungal without said second genetic modification. 9. The composition of claim 115, wherein the genetic modification results in increased expression of a gene product encoded by any one of PsiD, PsiH, PsiK, PsiTl, PsiT2, or PsiP. 0. The composition of claim 115, wherein the genetic modification results in an increased expression of the helix-loop-helix transcription factor as compared to a comparable fungal cell without the genetic modification. 1. The composition of claim 115, wherein the genetic modification results in an increased production of an alkaloid as compared to a comparable fungal cell without the genetic modification, wherein the alkaloid is a compound selected from psilocybin,

364 psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, and a salt thereof. . The composition of claim 115, wherein the genetically modified fungal cell is from division Basidiomycota. . The composition of claim 115, wherein the genetically modified fungal cell is a mycelium. . The composition of claim 115, wherein the genetically modified fungal cell is from Psilocybe, Conocybe, Gymnopilus, Panaeolus, Pluteus, and Stropharia. . The composition of claim 115, wherein the genetically modified fungal cell further comprises a modification that comprises a heterologous PsiH2 gene encoding a tryptamine monooxygenase. . The composition of claim 125, wherein the heterologous PsiH2 gene comprises a sequence that has 95% identity to one of SEQ ID NOS: 15 or 16. . The composition of claim 125, wherein the genetically modified fungal cell expresses an alkaloid or a precursor thereof that is not expressed in a detectable amount in a comparable organism without the first and second genetic modifications. . The composition of claim 102, wherein the alkaloid is a compound selected from

N,N-dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-Z-tryptophan, 5- hydroxy-Z-tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan- 1 -aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, harmine, harmane, P- carboline, and a pharmaceutically acceptable salt thereof. . The composition of claim 125, wherein the genetic modification and the modification result in increased expression of an alkaloid as compared to a comparable fungal cell without said modifications. . The fungal cell of any one of claims 115-129, wherein the genetic modification is accomplished by contacting a fungal cell with a gene editing system. . The fungal cell of any one of claims 115-129, wherein the genetic modification is accomplished by an agrobacterium-mediated insertion of the exogenous sequence. . The fungal cell of claim 117, wherein the gene editing system comprises one of CRISPR enzyme, TALE-Nuclease, transposon-based nuclease, Zinc finger nuclease, meganuclease, Mega-TAL or DNA guided nuclease.

365

. A pharmaceutical composition comprising the fungal cell or an extract of the fungal cell of any one of claims 115-119. . The pharmaceutical composition of claim 133, wherein the composition comprises an effective amount of the fungal cell or an extract of the fungal cell for treating a health condition. . The pharmaceutical composition of claim 133, wherein the composition is formulated such that an effective amount of the composition for treatment of the health condition can be delivered in a single dose format. . A nutraceutical composition comprising an extract of the genetically modified fungal cell of any one of claims 115-119. . A food supplement comprising an extract of the fungal cell of any one of claims 115-119. . A method of treatment, wherein the method comprises administering the fungal cell or an extract of the fungal cell of any one of claims 115-119 to a subject diagnosed with a health condition. . The method of claim 138, wherein the health condition is depression, anxiety, post- traumatic stress, addiction, or psychological distress including cancer-related psychological distress. . A composition comprising: a genetically modified fungal cell comprising: a first genetic modification that results in an increased expression of a heterologous tryptamine monooxygenase as compared to a comparable fungal cell without the first genetic modification; and a second genetic modification that results in an increased expression of a helix-loop-helix transcription factor as compared to a comparable fungal cell without the second genetic modification, wherein the helix-loop-helix transcription factor binds to a regulatory element of one or more genes that encode products of an alkaloid biosynthesis pathway. . The composition of claim 140, wherein the genetically modified fungal cell is from division Basidiomycota. . The composition of claim 128, wherein the heterologous tryptamine monooxygenase is homologous to a tryptamine monooxygenase that naturally occurs in a fungal cell from one of Psilocybe cyanescens, Psilocybe azurescence, or Psilocybe tampanensis.

366

. The composition of claim 140, wherein the heterologous tryptamine monooxygenase is encoded by a gene comprising a nucleotide sequence that is at least 95% identical to one of SEQ ID NOS: 15 or 16. . The composition of claim 140, wherein the helix-loop-helix transcription factor is encoded by a gene that comprises a nucleotide sequence that is at least 95% identical to one of SEQ ID NOS: 7 or 14. . The composition of claim 140, wherein the regulatory element comprises a hexanucleotide sequence that is an E-box motif. . The composition of claim 140, wherein the one or more genes comprise PsiD, PsiH, PsiM, PsiTl, PsiT2, or PsiP. . The composition of claim 146, wherein the helix-loop-helix transcription factor upregulates expression of the one or more genes as compared to a comparable fungal cell without the second genetic modification. . The composition of claim 140, wherein the genetically modified fungal cell produces an alkaloid that is not naturally expressed at a detectable amount in a comparable fungal cell without the first and second genetic modifications. . The composition of claim 140, wherein the genetically modified fungal cell is from Psilocybe cubensis. . The composition of claim 140, wherein the heterologous tryptamine monooxygenase is encoded by an exogenous nucleic acid. . The composition of claim 140, wherein the second genetic modification results in upregulated expression of any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, or a psilocybin-related phosphotransferase, as compared to a comparable organism without said second genetic modification. . The composition of claim 140, further comprising a modification that results in upregulated expression of at least one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, or a psilocybin-related phosphotransferase. . The composition of claim 140, wherein the first and second genetic modifications result in an increased production of an alkaloid as compared to a comparable fungal cell without the first and second genetic modifications, wherein the alkaloid is a compound selected from psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N- dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, N-acetyl-

367 hydroxytryptamine, 4-hydroxy-A-try ptophan, 5 -hydroxy -/.-tryptophan, 7-hydroxy-/,- tryptophan, 4-phosporyloxy-N,N-dimethyltryptamine, serotonin, aeruginascin, 2-(4- Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium, 4-phosphoryloxy-N,N- dimethytryptamine, ketamine, melatonin, normelatonin, 3,4- Methylenedioxymethamphetamine, harmine, harmane, P-carboline, and a salt thereof.. The composition of claim 140, wherein the fungal cell is a mycelium. . The genetically modified fungal cell of any one of claims 140-154, wherein the first genetic modification or the second genetic modification is accomplished by contacting a fungal cell with a gene editing system. . The genetically modified fungal cell of any one of claims 140-154, wherein the first genetic modification or the second genetic modification is accomplished by an agrobacterium-mediated insertion of the exogenous sequence. . The composition of claim 155, wherein the gene editing system comprises one of CRISPR enzyme, TALE-Nuclease, transposon-based nuclease, Zinc finger nuclease, meganuclease, Mega-TAL or DNA guided nuclease. . A pharmaceutical composition comprising the fungal cell or an extract of the fungal cell of any one of claims 140-157. . The pharmaceutical composition of claim 158, wherein the composition comprises an effective amount of the fungal cell or an extract of the fungal cell for treating a health condition. . The pharmaceutical composition of claim 159, wherein the composition is formulated such that an effective amount of the composition for treatment of the health condition can be delivered in a single dose format. . A nutraceutical composition comprising an extract of the fungal cell of any one of claims 140-157. . A food supplement comprising an extract of the fungal cell of any one of claims 140-157. . A method of treatment, wherein the method comprises administering the fungal cell or an extract of the fungal cell of any one of claims 140-157 to a subject diagnosed with a health condition. . The method of claim 163, wherein the health condition comprises one of depression, anxiety, post-traumatic stress, addiction, or psychological distress including cancer-related psychological distress. . A method of producing an alkaloid, the method comprising:

368 introducing a genetic modification into a fungal cell, wherein the genetic modification comprises an exogenous nucleic acid that encodes a tryptamine monooxygenase that is heterologous to the fungal cell; and expressing the tryptamine monooxygenase in the fungal cell to produce the alkaloid.

166. The method of claim 165, wherein the tryptamine monooxygenase is encoded by a PsiH2 gene comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO: 15 or 16.

167. The method of claim 165, wherein the exogenous nucleic acid is introduced into the fungal cell by an agrobacterium.

168. The method of claim 165, wherein the fungal cell is a fungal mycelium.

169. The method of claim 165, wherein the fungal cell is from division Basidiomycota.

170. The method of claim 165, wherein the heterologous tryptamine monooxygenase is homologous to a tryptamine monooxygenase of a fungus from one of Psilocybe cyanescens, Psilocybe azurescence, or Psilocybe tampanensis.

171. The method of claim 165, wherein the alkaloid that is produced is not expressed in a detectable amount in a comparable fungus without the genetic modification.

172. The method of claim 171, wherein the alkaloid is a compound selected from N,N- dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-Z-tryptophan, 5-hydroxy-/.- tryptophan, 7-hydroxy-Z-tryptophan, 4-phosporyloxy-N,N-dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy- lH-indol-3 -yl)-N,N,N-trimethylethan- 1 -aminium, 4- phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3,4- Methylenedioxymethamphetamine, harmine, harmane, P-carboline and a pharmaceutically acceptable salt thereof.

173. The method of claim 165, wherein the genetic modification results in increased expression of the alkaloid as compared to a fungus cell without the genetic modification.

174. The method of claim 173, wherein the alkaloid is at least one compound selected from psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, and a derivative or pharmaceutically acceptable salt thereof.

175. The method of claim 165, further comprising introducing a second genetic modification into the fungal cell, wherein the second genetic modification results in increased expression of a helix-loop-helix transcription factor that binds to a sequence comprising an E-box motif as compared to a comparable fungal cell without said second genetic modification.

369

176. The method of claim 175, wherein the second genetic modification is accomplished with a gene editing system.

177. The method of claim 176, wherein the gene editing system comprises any one of a CRISPR enzyme, TALE-Nuclease, transposon-based nuclease, Zinc finger nuclease, meganuclease, Mega-TAL or DNA guided nuclease.

178. A method of producing an alkaloid, the method comprising: introducing a genetic modification into a fungal cell, wherein the genetic modification comprises an exogenous nucleic acid that encodes a tryptamine monooxygenase that is heterologous to the fungal cell; expressing the tryptamine monooxygenase in the fungal cell to produce the alkaloid; and wherein the alkaloid comprises a compound of Formula (I):

-R¹ is H or -CH₃;

-R³ is H, -OH, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

-R⁴ and R⁵ are each independently H, -OH, or -OCH₃;

-R^A is H, -COOH;

179. The method of claim 178, wherein the tryptamine monooxygenase is encoded by a PsiH2 gene comprising a nucleotide sequence that is at least 95% identical to SEQ ID NO: 15 or 16.

180. The method of claim 178, wherein the exogenous nucleic acid is introduced into the fungal cell by an agrobacterium.

181. The method of claim 178, wherein the fungal cell comprises a mycelium.

182. The method of claim 178, wherein the fungal cell is from division Basidiomycota.

370

. The method of claim 178, wherein the heterologous tryptamine monooxygenase is homologous to a tryptamine monooxygenase of a fungus from one of Psilocybe cyanescens, Psilocybe azurescence, or Psilocybe tampanensis. . The method of claim 178, wherein the alkaloid that is produced is not expressed in a detectable amount in a comparable fungus without the genetic modification. . The method of claim 178, wherein the alkaloid is a compound according to formulae (la), (lb), and (Ic). . The method of claim 178, wherein the alkaloid is a compound selected from compounds 1-19. . The method of claim 178, wherein the alkaloid is a non-naturally occurring compound. . The method of claim 178, wherein the alkaloid is an N-methylated derivative of any one of compounds 1-19. . The method of claim 178, further comprising introducing a second genetic modification into the fungal cell, wherein the second genetic modification results in increased expression of a helix-loop-helix transcription factor that binds to a sequence comprising SEQ ID NO: 7 or 14 as compared to a comparable fungal cell without said second genetic modification. . The method of claim 178, wherein the alkaloid is formulated in an effective amount for treatment of a health condition. . The method of any one of claims 178-190, wherein the alkaloid is formulated in an effective amount for treatment of a health condition. . The method of any one of claims 178-190, wherein the alkaloid is formulated in an effective amount for treatment of a health condition in single dose format. . The method of any one of claims 178-190, wherein the alkaloid is formulated as a pharmaceutical composition in an effective amount for treatment of a health condition, wherein the health condition is depression, anxiety, post-traumatic stress, addiction, or psychological distress including cancer-related psychological distress. . The method of any one of claims 178-190, wherein the alkaloid is formulated in a pharmaceutical composition in an effective amount for treatment of a health condition, wherein the composition further comprises a pharmaceutically acceptable carrier. . The method of any one of claims 178-190, wherein the alkaloid is formulated in an effective amount for use as a supplement.

371

. The method of any one of claims 178-190, wherein the alkaloid is formulated in an effective amount for use as a nutraceutical composition. . The method of any one of claims 178-190, wherein the alkaloid is formulated in an effective amount for use as a food supplement. . The method of claim 189, wherein the second genetic modification is accomplished with a gene editing system. . The method of claim 185, wherein the gene editing system comprises any one of a CRISPR enzyme, TALE-Nuclease, transposon-based nuclease, Zinc finger nuclease, meganuclease, Mega-TAL or DNA guided nuclease. . A composition comprising:

R³ is H, -OH, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

R⁴ and R⁵ are each independently H, -OH, or -OCH₃;

R^A is H, -COOH;

R^x, R^y, and R^z are each independently H, -CH₃, -C(O)CH₃, -CH₂CH₃, or -CH(CH₃)₂; and

M is H, Li⁺, Na⁺, K⁺ or is absent; and material from a ruptured fungal cell. . The composition of claim 200, wherein the material comprises a nucleic acid. . The composition of claim 201, wherein the nucleic acid comprises a sequence that has at least a 95% identity to one of SEQ ID NOS: 15 or 16. . The composition of claim 200, wherein the fungal cell is from division Basidiomycota. . A composition comprising:

372 a genetically modified organism comprising an exogenous nucleic acid, wherein the exogenous nucleic acid encodes a heterologous methyl transferase that is not expressed in a detectable amount in a comparable organism without said exogenous nucleic acid.. The composition of claim 204, wherein the heterologous methyl transferase is encoded by a fungal PsiM gene. . The composition of claim 205, wherein the PsiM gene is not present in the comparable organism. . The composition of claim 204, wherein the genetically modified organism is a multicellular organism. . The composition of claim 204, wherein the multicellular organism comprises a fungus. . The composition of claim 204, wherein the fungus is from division Basidiomycota.. The composition of claim 204, wherein the heterologous methyl transferase comprises a methyl transferase from Psilocybe azurescence. . The composition of claim 195, wherein the fungus is from Psilocybe cubensis and wherein the methyl transferase is from Psilocybe azurescence. . The composition of claim 204, wherein, when the methyl transferase is expressed, the genetically modified organism produces an alkaloid. . The composition of claim 212, wherein the alkaloid is a compound selected from

N,N-dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-Z-tryptophan, 5- hydroxy-Z-tryptophan, 7-hydroxy-Z-tryptophan, isonorbaeocystin, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan- 1 -aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, harmine, harmane, P- carboline, and a salt thereof. . The composition of claim 204, wherein the exogenous nucleic acid results in increased expression of an alkaloid as compared to a comparable organism without the genetic modification. . The composition of claim 214, wherein the alkaloid is a compound according to Formula (I):

373

-R¹ is H or -CH₃;

-R³ is H, -OH, -OCH₃, -OCH₂CH₃, or -OCH(CH₃)₂;

-R⁴ and R⁵ are each independently H, -OH, or -OCH₃;

-R^A is H, -COOH;

216. The composition of claim 214, wherein the alkaloid is a compound selected from psilocybin, psilocin, tryptamine, 4-hydroxytryptamine, N,N-dimethyltryptamine, norpsilocin, baeocystin, norbaeocystin, isonorbaeocystin, isonorbaeocystin, and a salt thereof.

217. A composition comprising: a fungal cell comprising a genetic modification that results in the fungal cell expressing a heterologous PsiM gene that is not expressed in a detectable amount in a comparable fungal cell without said genetic modification.

218. The composition of claim 217, wherein the PsiM gene is from Psilocybe Azurescence.

219. A composition comprising: a fungal cell comprising a genetic modification that results in the fungal cell expressing a heterologous TrpM gene that is not expressed in a detectable amount in a comparable fungal cell without said genetic modification.

220. The composition of claim 219, wherein the PsiM gene is from Psilocybe Azurescence.

221. A gene editing system for enhanced expression of a psychotropic alkaloid in a fungal cell, wherein the system comprises:

374 an endonuclease and at least one guide polynucleotide, or one or more nucleic acids encoding the endonuclease and the at least one guide polynucleotide, wherein the guide polynucleotide binds to a nucleic acid that encodes or regulates a gene that modulates production of a psychotropic alkaloid in a fungal cell; and a reagent that enhances incorporation of the endonuclease or the at least one guide polynucleotide, or the one or more nucleic acids encoding the endonuclease or the at least one guide polynucleotide, into the fungal cell as compared to the incorporation absent said reagent. . The system of claim 221, wherein the fungal cell is a fungal protoplast. . The system of claim 221, wherein the fungal cell is from the genus Psilocybe.. The system of claim 221, wherein the system comprises the endonuclease and the at least one guide polynucleotide in a single composition. . The system of claim 221, wherein the endonuclease and the at least one guide polynucleotide are in separate compositions. . The system of claim 221, wherein the system comprises the one or more nucleic acids encoding the endonuclease and the at least one guide polynucleotide. . The system of claim 221, wherein the endonuclease and the at least one guide polynucleotide are encoded on a single nucleic acid. . The system of claim 227, wherein the single nucleic acid comprises a nonreplicating vector. . The system of claim 221, wherein the endonuclease and the at least one guide polynucleotide are encoded on different nucleic acids. . The system of claim 221, wherein the one or more nucleic acids comprises a gene promoter that drives expression of the endonuclease or the at least one guide polynucleotide in the fungal cell. . The system of claim 230, wherein the gene promoter comprises a sequence that has at least 95 percent identity to one of SEQ ID NOS: 88-90, or 93. . The system of claim 221, wherein the one or more nucleic acids comprise a first gene promoter that drives expression of the endonuclease and a second gene promoter that drives expression of the at least one guide polynucleotide, wherein the first gene protomer is different from the second gene promoter. . The system of claim 232, wherein the first gene promoter is a GDP gene promoter, and the second gene promoter is a U6 gene promoter.

375

. The system of claim 221, wherein the endonuclease is a complex with the at least one guide polynucleotide and the reagent enhances the incorporation of the complex into the fungal cell. . The system of claim 221, wherein the gene comprises a psilocybin synthase gene.. The system of claim 221, wherein the gene comprises one of tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif. . The system of claim 221, wherein the gene comprises a sequence that has at least 75% identity to one of SEQ ID NOS: 1-18. . The system of claim 221, wherein the at least one guide polynucleotide can bind to a target sequence that comprises at least 95 percent identity to one of SEQ ID NOS: 31-87. . The system of claim 221, wherein the system comprises at least two guide polynucleotides. . The system of claim 221, wherein the reagent comprises a nonionic surfactant, a lipid nanoparticle, a detergent, or an agent that depolymerizes microtubules. . The system of claim 221, wherein the reagent is Triton X-100. . The system of claim 221, wherein the reagent comprises inositol or benomyl.. The system of claim 221, wherein the system further comprises an exogenous nucleic acid. . The system of claim 243, wherein the exogenous nucleic acid comprises a psilocybin synthase gene. . The system of claim 243, wherein the exogenous nucleic acid comprises any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif. . The system of claim 243, wherein the exogenous nucleic acid comprises a sequence that has at least a 95 percent identity to one of SEQ ID NOS: 1-18. . The system of claim 243, wherein the exogenous nucleic acid results in the increased expression of a polypeptide comprising a sequence that has at least 95 percent identity to one of SEQ ID NOS: 19-30.

376

. The system of claim 221, wherein the endonuclease is a Cas endonuclease, a Cas endonuclease variant, or an Argonaut. . The system of claim 221, wherein the endonuclease is selected from a group consisting of Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Cpfl, c2cl, c2c3, nCas, and Cas9HiFi. . The system of claim 221, wherein the endonuclease comprises a nuclear localization signal. . The system of claim 250, wherein the nuclear localization signal is encoded by a nucleic acid sequence optimized for expression in a fungus from the Psilocybe genus.. The system of claim 248, wherein the Cas endonuclease is encoded by a nucleic acid sequence optimized for expression in a fungus from the Psilocybe genus. . The system of claim 252, wherein the polynucleotide comprises codons that frequently occur in the Psilocybe genus. . The system of claim 221, wherein the at least one guide polynucleotide comprises a guide DNA or a guide RNA. . The system of claim 254, wherein the at least one guide polynucleotide is guide RNA. . The system of claim 221, wherein expression of the endonuclease and the at least one guide polynucleotide inside the fungal cell results in a genetic modification that leads to an increased expression of the psychotropic alkaloid as compared to a comparable fungal cell without the endonuclease and the at least one guide RNA, or the one or more nucleic acids encoding the endonuclease and the at least one guide RNA. . The system of claim 256, wherein the psychotropic alkaloid is a compound selected from N,N-dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-L- tryptophan, 5 -hydroxy -L-tryptophan, 7-hydroxy-L-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan- 1 -aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, harmine, harmane, and P-carboline. . A gene editing system for genetically modifying a fungal cell, wherein the system comprises:

377 an endonuclease and at least one guide polynucleotide, or one or more nucleic acids encoding said endonuclease and the at least one guide polynucleotide, wherein the guide polynucleotide can bind to a gene that comprises a sequence comprising one of SEQ ID NOS: 31-87, wherein, binding of the guide polynucleotide to the gene in a fungal cell leads to a genetic modification that modulates production of one or more alkaloids.. The system of claim 258, wherein the fungal cell is from division Basidiomycota.. The system of claim 258, wherein the fungal cell is a fungal protoplast. . The system of claim 258, wherein the at least one guide polynucleotide binds to the gene within 100 bases of the sequence comprising one of SEQ ID NOS: 31-87. . The system of claim 258, wherein the at least one guide polynucleotide at least partially binds to the sequence of one of SEQ ID NOS: 31-87. . The system of claim 258, wherein the at least one guide polynucleotide comprises a targeting sequence that is complementary to one of SEQ ID NOS: 31-87. . The system of claim 258, wherein the at least one guide polynucleotide comprises a targeting sequence that binds to one of SEQ ID NOS: 31-87. . The system of claim 258, wherein the system comprises a polynucleotide comprises a targeting sequence that binds to one of SEQ ID NOS: 31-87. . The system of claim 258, wherein the system comprises the endonuclease and the at least one guide polynucleotide in a single composition. . The system of claim 258, wherein the endonuclease and the at least one guide polynucleotide are in separate compositions. . The system of claim 258, wherein the gene comprises any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif. . The system of claim 258, wherein the gene comprises any one of PsiD, PsiH, PsiH2, PsiK, PsiM, PsiTl, PsiT2, PsiR, and PsiP. . The system of claim 258, wherein the system comprises the one or more nucleic acids encoding the endonuclease and the at least one guide polynucleotide. . The system of claim 270, wherein the endonuclease and the at least one guide polynucleotide are encoded on a single nucleic acid. . The system of claim 271, wherein the single nucleic acid comprises a nonreplicating vector.

378

. The system of claim 270, wherein the endonuclease and the at least one guide polynucleotide are encoded on different nucleic acids. . The system of claim 270, wherein the one or more nucleic acids comprises a gene promoter that drives expression of the endonuclease or the at least one guide polynucleotide in the fungal cell. . The system of claim 274, wherein the gene promoter comprises a sequence that comprises at least 95 percent identity to one of SEQ ID NOS: 88-90, or 93. . The system of claim 270, wherein the one or more nucleic acids comprise a first gene promoter that drives expression of the endonuclease and a second gene promoter that drives expression of the at least one guide polynucleotide, wherein the first gene protomer is different from the second gene promoter. . The system of claim 276, wherein the first gene promoter comprises a GDP gene promoter and the second gene promoter comprises a U6 gene promoter. . The system of claim 258, wherein the endonuclease is in a complex with the at least one guide polynucleotide. . The system of claim 258, wherein the gene comprises tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif. . The system of claim 258, wherein the system comprises at least two guide polynucleotides. . The system of claim 258, wherein the system further comprises an exogenous nucleic acid. . The system of claim 281, wherein the exogenous nucleic acid comprises a psilocybin synthase gene. . The system of claim 281, wherein the exogenous nucleic acid comprises any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif. . The system of claim 258, wherein the endonuclease is a Cas endonuclease, a Cas endonuclease variant, or an Argonaut. . The system of claim 258, wherein the endonuclease is selected from a group consisting of Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9, CaslO,

379 Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Cpfl, c2cl, c2c3, and Cas9HiFi. . The system of claim 284, wherein the Cas endonuclease is a Cas9 endonuclease.. The system of claim 284, wherein the endonuclease is encoded by a polynucleotide optimized for expression in the Psilocybe genus by comprising codons that frequently occur in the Psilocybe genus. . The system of claim 258, wherein the endonuclease comprises a nuclear localization signal. . The system of claim 258, wherein the at least one guide polynucleotide comprises a guide RNA. . The system of claim 258, wherein expression of the endonuclease and the at least one guide RNA inside the fungal cell results in a genetic modification that leads to an increased expression of the psychotropic alkaloid as compared to a comparable wild type fungal cell. . The system of claim 258, wherein the psychotropic alkaloid is a compound selected from N,N-dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-L- tryptophan, 5 -hydroxy -L-tryptophan, 7-hydroxy-L-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan- 1 -aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, harmine, harmane, and P-carboline. . The system of claim 258, wherein the system comprises the endonuclease and the at least one guide polynucleotide in a single composition. . The system of claim 258, wherein the endonuclease and the at least one guide polynucleotide are in separate compositions. . The system of claim 258, wherein the system comprises the one or more nucleic acids encoding the endonuclease and the at least one guide polynucleotide. . A gene editing system for genetically modifying a fungal cell, the system comprising: at least one nucleic acid sequence encoding a guide polynucleotide that binds to a nucleic acid in a fungal cell, wherein the at least one nucleic acid sequence is operably linked to a first gene promoter; and

380 a nucleic acid sequence encoding an endonuclease operably linked to a second gene promoter, wherein the second gene promoter is distinct from the first gene promoter, wherein, when the gene editing system is expressed in a fungal cell, the gene editing system introduces a genetic modification into the genome of the fungal cell. . The system of claim 295, wherein the first gene promoter is a fungal gene promoter. . The system of claim 296, wherein the fungal gene promoter comprises a U6 gene promoter. . The system of claim 297, wherein the U6 gene promoter comprises a sequence that has at least 95 percent identity to one of SEQ ID NOS: 88-90. . The system of claim 295, wherein the second gene promoter comprises a GPD gene promoter from Agaricus bisporus. . The system of claim 295, wherein the guide polynucleotide binds to a gene that comprises a sequence comprising one of SEQ ID NOS: 31-87. . The system of claim 300, wherein the at least one guide polynucleotide binds to the gene within 100 bases of the sequence comprising one of SEQ ID NOS: 31-87. . The system of claim 300, wherein the at least one guide polynucleotide binds to the gene at least partially on the sequence comprising one of SEQ ID NOS: 31-87. . The system of claim 300, wherein the at least one guide polynucleotide comprises a targeting sequence that is complementary to one of SEQ ID NOS: 31-87. . The system of claim 300, wherein the at least one guide polynucleotide comprises a targeting sequence that binds to one of SEQ ID NOS: 31-87. . The system of claim 295, further comprising a fungal cell from division Basidiomycota. . The system of claim 301, wherein the fungal cell is a fungal protoplast. . The system of claim 295, wherein the system further comprises an exogenous nucleic acid. . The system of claim 307, wherein the exogenous nucleic acid comprises a psilocybin synthase gene. . The system of claim 308, wherein the exogenous nucleic acid comprises any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif.

381

. The system of claim 295, wherein the endonuclease is a Cas endonuclease. . The system of claim 310, wherein the Cas endonuclease is a Cas9 endonuclease.. The system of claim 311, wherein the endonuclease is encoded by a polynucleotide optimized for expression in the Psilocybe genus by comprising codons that frequently occur in the Psilocybe genus. . The system of claim 295, wherein the at least one guide polynucleotide comprises a guide RNA. . The system of claim 295, wherein expression of the at least one guide polynucleotide and Cas endonuclease inside the fungal cell results in a genetic modification that leads to an increased expression of the psychotropic alkaloid as compared to a comparable wild type fungal cell. . The system of claim 314, wherein the psychotropic alkaloid is a compound selected from N,N-dimethytryptamine, N-acetyl-hydroxytryptamine, 4-hydroxy-L- tryptophan, 5 -hydroxy -L-tryptophan, 7-hydroxy-L-tryptophan, 4-phosporyloxy-N,N- dimethyltryptamine, serotonin, aeruginascin, 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N- trimethylethan- 1 -aminium, 4-phosphoryloxy-N,N-dimethytryptamine, ketamine, melatonin, normelatonin, 3, 4-Methylenedi oxymethamphetamine, harmine, harmane, and P-carboline. . The system of claim 295, wherein the endonuclease comprises a nuclear localization signal. . The system of claim 295, wherein the system comprises the endonuclease and the at least one guide polynucleotide in a single composition. . The system of claim 295, wherein the endonuclease and the at least one guide polynucleotide are in separate compositions.. A gene editing system for multiplex gene engineering of a fungal cell, wherein the system comprises: a vector encoding: at least two guide polynucleotides that each bind to a nucleic acid that encodes or regulates a gene that modulates production of a psychotropic alkaloid in a fungal cell; and an endonuclease, wherein when the at least two guide polynucleotides and the endonuclease are expressed in a fungal cell, the at least two guide polynucleotides and the endonuclease introduce a genetic modification into the genome of the fungal cell.

382

. The system of claim 319, wherein the vector comprises a binary agrobacterium vector. . The system of claim 319, wherein vector further comprises a first border sequence and a second border sequence flanking the sequence encoding the at least two guide polynucleotides and the endonuclease, wherein the first border sequence and the second border sequence comprise a repeat that is associated with agrobacterium-mediated transformation. . The system of claim 320, wherein the first and second border sequences comprise a repeat. . The system of claim 319, wherein the vector comprises at least four guide polynucleotides. . The system of claim 319, wherein the at least two polynucleotide are positioned on the vector downstream of a fungal gene promoter. . The system of claim 324, wherein the fungal gene promoter is a U6 promoter comprising a sequence that has at least 95 percent identity to one of SEQ ID NOS: 88-90.. The system of claim 324, wherein the endonuclease is regulated by a second gene promoter that is different from the fungal gene promoter. . The system of claim 326, wherein the second gene promoter comprises a GPD gene promoter from Agaricus bisporus. . The system of claim 319, wherein the endonuclease comprises a Cas9 endonuclease, or a Cas9 variant, linked to a nuclear localization signal. . The system of claim 319, wherein the polynucleotide sequence encoding the endonuclease is optimized comprises amino acid codons that frequently occur in the Psilocybe genus. . The system of claim 319, wherein the nucleic acid involved in production or regulation of a psychotropic alkaloid comprises a sequence that encodes any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif. . The system of claim 319, wherein the nucleic acid involved in production or regulation of a psychotropic alkaloid comprises a sequence that encodes any one of PsiD, PsiH, PsiH2, PsiK, PsiM, PsiTl, PsiT2, PsiR, and PsiP.

383

. The system of claim 319, wherein the gene comprises a sequence comprising at least one of SEQ ID NOS: 31-87. . The system of claim 332, wherein the at least one guide polynucleotide binds to the gene within 100 bases of the sequence comprising one of SEQ ID NOS: 31-87. . The system of claim 332, wherein the at least one guide polynucleotide binds to the gene at least partially on the sequence comprising one of SEQ ID NOS: 31-87. . The system of claim 332, wherein the at least one guide polynucleotide comprises a targeting sequence that is complementary to one of SEQ ID NOS: 31-87. . The system of claim 332, wherein the at least one guide polynucleotide comprises a targeting sequence that binds to one of SEQ ID NOS: 31-87. . The system of claim 332, wherein one of the at least two guide polynucleotides comprises a sequence that can bind to a target sequence comprising one of SEQ ID NOS: 31-87. . The system of claim 319, wherein, when the at least two guide polynucleotides and the endonuclease is expressed in a fungal cell the at least two guide polynucleotides and the endonuclease results in increased expression of any one of a tryptophan decarboxylase, a psilocybin hydroxylase, a psilocybin-related methyltransferase, a psilocybin-related kinase, a psilocybin-related phosphotransferase, or helix-loop-helix transcription factor that binds to an E-box motif, as compared to a comparable wild type fungal cell. . The system of claim 319, wherein one of the at least two guide polynucleotides comprises a sequence that binds to a promoter or an enhancer of a psilocybin biosynthesis gene. . The system of claim 339, wherein the psilocybin biosynthesis gene is selected from the group consisting of: PsiD, PsiK, PsiH, PsiM, PsiR, PsiP2, PsiPl, TrpE, and TrpM. . The system of claim 319, wherein the system further comprises an exogenous nucleic acid. . The system of claim 341, wherein the exogenous nucleic acid comprises a psilocybin synthase gene. . The system of claim 341, wherein the exogenous nucleic acid comprises any one of a tryptophan decarboxylase gene, a psilocybin hydroxylase gene, a psilocybin-related methyltransferase gene, a psilocybin-related kinase gene, a psilocybin-related

384 phosphotransferase gene, or a gene encoding a helix-loop-helix transcription factor that binds to an E-box motif. . The system of claim 341, wherein the exogenous nucleic acid comprises a sequence that has at least 95 percent identity to one of SEQ ID NOS: 1-18. . The system of claim 341, wherein the exogenous nucleic acid results in the increased expression of a polypeptide comprising a sequence that has at least 95 percent identity to one of SEQ ID NOS: 19-30. . A kit for genetically modifying a fungal cell, the kit comprising: a gene editing system of any one of claims 1-345; and

Triton X-100. . A method for making a genetic modification, the method comprising: introducing the gene editing system of any one of claims 221-345 into a fungal cell. . The method of claim 347, further comprising expressing the gene editing system in the fungal cell. . The method of claim 348, wherein the fungal cell is from division Basidiomycota. The method of claim 348, wherein the endonuclease is pre-complexed with the guide polynucleotide. . The method of claim 348, wherein the gene editing system is delivered as a vector into the fungal cell. . The method of claim 348, wherein the gene editing system is introduced into the fungal cell with a bacterium. . The method of claim 352, wherein the bacterium is Agrobacterium tumefaciens.. The method of claim 348, wherein expression of the gene editing system in the fungal cell results in any one of increased tryptophan decarboxylation, increased tryptamine 4-hydroxylation, increased 4-hydroxytryptamine O-phosphorylation, or increased psilocybin production via sequential N-methylations in the fungal cell as compared to a comparable fungal cell without the gene editing system. . The method of claim 348, wherein the expression of the gene editing system results in increased production of an alkaloid by the fungal cell as compared to a comparable fungal cell without the gene editing system, wherein the alkaloid comprises a compound selected from N,N-dimethytryptamine, N-acetyl-hydroxytryptamine, 4- hydroxy-L-tryptophan, 5 -hydroxy -L-tryptophan, 7-hydroxy-L-tryptophan, 4- phosporyloxy-N,N-dimethyltryptamine , serotonin or a derivative thereof, aeruginascin ,

385 2-(4-Hydroxy-lH-indol-3-yl)-N,N,N-trimethylethan-l-aminium , 4-phosphoryloxy-N,N- dimethytryptamine, ketamine, melatonin or a derivative thereof, normelatonin, 3,4- Methylenedioxymethamphetamine, isatin, harmine, P-carboline, N,N-dimethyltryptamine (DMT) or a derivative thereof. . The system or method of any preceding claim, further comprising a reagent that comprises at least one of a nonionic surfactant, a lipid nanoparticle, a detergent, or an agent that depolymerizes microtubules. . The system or method of any preceding cl im, further comprising Triton X-100.. The system or method of any preceding claim, further comprising inositol or benomyl. . A pharmaceutical composition comprising the composition of any preceding claim, and a pharmaceutically acceptable: diluent, carrier, excipient, or any combination thereof. . The pharmaceutical composition of claim 359, that is in unit dose form. . A kit comprising the pharmaceutical composition of any preceding claim, and a container. . A method of treating a health condition, disease, or disorder in a subject, the method comprising administering the composition or pharmaceutical composition of any preceding claim, to the subject in an amount effective to treat the health condition, disease, or disorder in the subject. . The method of claim 362, wherein the subject is a subject in need thereof. . The method of claim 362 or claim 363, wherein the health condition, disease or disorder is a neurological health condition, disease, or disorder. . The method of claim 364, wherein the neurological health condition, disease, or disorder is: a depression, an anxiety, a post-traumatic stress disorder (PTSD), a psychiatric disorder, mental trauma, a mood disorder, a speech disorder, neurodegenerative disease, psychological distress, a compulsion, a compulsive disorder, an obsessive disorder, an expression of a symptom in a neurodivergent individual, cancer-related psychological distress, an addiction, a headache, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease a phobia, a dementia, a fear, an eating disorder, an ischemic event, or any combination thereof.. The composition or pharmaceutical composition of any preceding claim, further comprising a monoamine oxidase (MAO) inhibitor, an inhibitor of MAO A, or an inhibitor of MAO B.

386

. The method of any preceding claim, further comprising administering concurrently or consecutively with the composition or pharmaceutical composition, a monoamine oxidase (MAO) inhibitor, an inhibitor of MAO A, or an inhibitor of MAO B.

387